RESUMEN
The ultimate goal in radiation oncology is to offer a personalized treatment to all patients indicated for radiotherapy. Radiomics is a tool that reinforces a deep analysis of tumors at the molecular aspect taking into account intrinsic susceptibility in a long-term follow-up. Radiomics allow qualitative and quantitative performance analyses with high throughput extraction of numeric radiologic data to obtain predictive or prognostic information from patients treated for cancer. A second approach is to define biological or constitutional that could change the practice. This technique included normal tissue individual susceptibility but also potential response of tumors under ionizing radiation treatment. These "omics" are biological and technical techniques leading to simultaneous novel identification and exploration a set of genes, lipids, proteins.
Asunto(s)
Radioterapia/métodos , Genómica , Humanos , Neoplasias/radioterapia , Proteómica , Radiación IonizanteRESUMEN
Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-ß-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.
Asunto(s)
Imidazoles/farmacología , Necrosis/tratamiento farmacológico , Piridazinas/farmacología , Pirimidinas/farmacología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Sulfonamidas/farmacología , Células 3T3 , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proteína de Dominio de Muerte Asociada a Fas/genética , Células HEK293 , Células HT29 , Humanos , Indazoles , Células Jurkat , Células L , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Breast cancer is genetically heterogeneous, and recent studies have underlined a prominent contribution of epigenetics to the development of this disease. To uncover new synthetic lethalities with known breast cancer oncogenes, we screened an epigenome-focused short hairpin RNA library on a panel of engineered breast epithelial cell lines. Here we report a selective interaction between the NOTCH1 signaling pathway and the SUMOylation cascade. Knockdown of the E2-conjugating enzyme UBC9 (UBE2I) as well as inhibition of the E1-activating complex SAE1/UBA2 using ginkgolic acid impairs the growth of NOTCH1-activated breast epithelial cells. We show that upon inhibition of SUMOylation NOTCH1-activated cells proceed slower through the cell cycle and ultimately enter apoptosis. Mechanistically, activation of NOTCH1 signaling depletes the pool of unconjugated small ubiquitin-like modifier 1 (SUMO1) and SUMO2/3 leading to increased sensitivity to perturbation of the SUMOylation cascade. Depletion of unconjugated SUMO correlates with sensitivity to inhibition of SUMOylation also in patient-derived breast cancer cell lines with constitutive NOTCH pathway activation. Our investigation suggests that SUMOylation cascade inhibitors should be further explored as targeted treatment for NOTCH-driven breast cancer.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Receptor Notch1/genética , Transducción de Señal/genética , Activación Transcripcional , Apoptosis/efectos de los fármacos , Apoptosis/genética , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Técnicas de Cocultivo , Citometría de Flujo , Humanos , Microscopía Fluorescente , Interferencia de ARN , Receptor Notch1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Salicilatos/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación/efectos de los fármacos , Sumoilación/genética , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
The microphthalmia-associated transcription factor (MITF) is indispensable for the viability of melanocytic cells, is an oncogene in melanoma and has a cell type-specific expression pattern. As the modulation of MITF activity by direct chemical targeting remains a challenge, we assessed a panel of drugs for their ability to downregulate MITF expression or activity by targeting its upstream modulators. We found that the multi-kinase inhibitors midostaurin and sunitinib downregulate MITF protein levels. To identify the target molecules shared by both the drugs in melanocytic cells, a chemical proteomic approach was applied and AMP-activated kinase (AMPK) was identified as the relevant target for the observed phenotype. RNA interference and chemical inhibition of AMPK led to a decrease in MITF protein levels. Reduction of MITF protein levels was the result of proteasomal degradation, which was preceded by enhanced phosphorylation of MITF mediated by ERK. As expected, downregulation of MITF protein levels by AMPK inhibition was associated with decreased viability. Together, these results identify AMPK as an important regulator for the maintenance of MITF protein levels in melanocytic cells.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Melanocitos/metabolismo , Melanoma/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Western Blotting , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cromatografía Liquida , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Indoles/farmacología , Espectrometría de Masas , Melanocitos/efectos de los fármacos , Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/genética , Oncogenes , Pirroles/farmacología , Interferencia de ARN , ARN Interferente Pequeño , Estaurosporina/análogos & derivados , Estaurosporina/farmacología , Sunitinib , TransfecciónRESUMEN
Resistance to the BCR-ABL tyrosine kinase inhibitor imatinib poses a pressing challenge in treating chronic myeloid leukemia (CML). This resistance is often caused by point mutations in the ABL kinase domain or by overexpression of LYN. The second-generation BCR-ABL inhibitor INNO-406 is known to inhibit most BCR-ABL mutants and LYN efficiently. Knowledge of its full target spectrum would provide the molecular basis for potential side effects or suggest novel therapeutic applications and possible combination therapies. We have performed an unbiased chemical proteomics native target profile of INNO-406 in CML cells combined with functional assays using 272 recombinant kinases thereby identifying several new INNO-406 targets. These include the kinases ZAK, DDR1/2 and various ephrin receptors. The oxidoreductase NQO2, inhibited by both imatinib and nilotinib, is not a relevant target of INNO-406. Overall, INNO-406 has an improved activity over imatinib but a slightly broader target profile than both imatinib and nilotinib. In contrast to dasatinib and bosutinib, INNO-406 does not inhibit all SRC kinases and most TEC family kinases and is therefore expected to elicit fewer side effects. Altogether, these properties may make INNO-406 a valuable component in the drug arsenal against CML.
Asunto(s)
Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteómica , Pirimidinas/farmacología , Receptor con Dominio Discoidina 1 , Receptores con Dominio Discoidina , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Quinasas Quinasa Quinasa PAM , Proteínas Quinasas/fisiología , Quinona Reductasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/fisiología , Receptores Mitogénicos/antagonistas & inhibidoresRESUMEN
The detailed molecular mechanism of action of second-generation BCR-ABL tyrosine kinase inhibitors, including perturbed targets and pathways, should contribute to rationalized therapy in chronic myeloid leukemia (CML) or in other affected diseases. Here, we characterized the target profile of the dual SRC/ABL inhibitor bosutinib employing a two-tiered approach using chemical proteomics to identify natural binders in whole cell lysates of primary CML and K562 cells in parallel to in vitro kinase assays against a large recombinant kinase panel. The combined strategy resulted in a global survey of bosutinib targets comprised of over 45 novel tyrosine and serine/threonine kinases. We have found clear differences in the target patterns of bosutinib in primary CML cells versus the K562 cell line. A comparison of bosutinib with dasatinib across the whole kinase panel revealed overlapping, but distinct, inhibition profiles. Common among those were the SRC, ABL and TEC family kinases. Bosutinib did not inhibit KIT or platelet-derived growth factor receptor, but prominently targeted the apoptosis-linked STE20 kinases. Although in vivo bosutinib is inactive against ABL T315I, we found this clinically important mutant to be enzymatically inhibited in the mid-nanomolar range. Finally, bosutinib is the first kinase inhibitor shown to target CAMK2G, recently implicated in myeloid leukemia cell proliferation.
Asunto(s)
Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , Células K562/efectos de los fármacos , Leucemia Mieloide de Fase Acelerada/enzimología , Proteínas de Neoplasias/antagonistas & inhibidores , Nitrilos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinolinas/farmacología , Compuestos de Anilina/química , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Dasatinib , Sistemas de Liberación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Perfilación de la Expresión Génica , Humanos , Células K562/enzimología , Leucemia Mieloide de Fase Acelerada/patología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/enzimología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Nitrilos/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Pirimidinas/farmacología , Quinolinas/química , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato , Tiazoles/farmacología , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
UNLABELLED: SAGE data are obtained by sequencing short DNA tags. Due to the mistakes in DNA sequencing, SAGE data contain errors. We propose a new approach to identify tags whose abundance is biased by sequencing errors. This approach is based on a concept of neighbourhood: abundant tags can contaminate tags whose sequence is very close. The application of our approach reveals that moderately abundant tags can be generated by sequencing errors uniquely. It also allows for detecting correct rare tags. AVAILABILITY: Software is available only to non-profit entities and for non-commercial purposes upon request.
Asunto(s)
Perfilación de la Expresión Génica , Biblioteca de Genes , Análisis de Secuencia de ADN/métodos , Perfilación de la Expresión Génica/métodos , ARN Mensajero/aislamiento & purificaciónRESUMEN
At increasing speed, sequencing data are being made public from both complex and simple life forms. Although biomedical interests tend to focus on mammalian genes, only simple organisms allow rapid genetic manipulation and functional analysis. A prerequisite for the meaningful extrapolation of gene functional studies from invertebrates to man is that the orthologs under study are unambiguously linked. However, identifying orthologs is not trivial, especially where large gene families are involved. We present here an automated sequence analysis procedure that allows a rapid visualization of most likely ortholog pairs. We illustrate the utility of this approach for the human gene family of protein tyrosine phosphatases (PTPs) as compared with the full set of Caenorhabditis elegans and Drosophila melanogaster conceptual ORFs. The approach is based on a reciprocal series of BLAST searches, which are automatically stored and represented in an HTML-formatted table. We have used this 'MetaBlast' approach to compile lists of human PTPs and their worm and fly orthologs. Many of these PTP orthologs had not been previously identified as such.