RESUMEN
The BCR/ABL1 inhibitor Nilotinib is increasingly used to treat patients with chronic myeloid leukemia (CML). Although otherwise well-tolerated, Nilotinib has been associated with the occurrence of progressive arterial occlusive disease (AOD). Our objective was to determine the exact frequency of AOD and examine in vitro and in vivo effects of Nilotinib and Imatinib on endothelial cells to explain AOD-development. In contrast to Imatinib, Nilotinib was found to upregulate pro-atherogenic adhesion-proteins (ICAM-1, E-selectin, VCAM-1) on human endothelial cells. Nilotinib also suppressed endothelial cell proliferation, migration and tube-formation and bound to a distinct set of target-kinases, relevant to angiogenesis and atherosclerosis, including angiopoietin receptor-1 TEK, ABL-2, JAK1 and MAP-kinases. Nilotinib and siRNA against ABL-2 also suppressed KDR expression. In addition, Nilotinib augmented atherosclerosis in ApoE-/- mice and blocked reperfusion and angiogenesis in a hindlimb-ischemia model of arterial occlusion, whereas Imatinib showed no comparable effects. Clinically overt AOD-events were found to accumulate over time in Nilotinib-treated patients. After a median observation-time of 2.0 years, the AOD-frequency was higher in these patients (29.4%) compared to risk factor- and age-matched controls (<5%). Together, Nilotinib exerts direct pro-atherogenic and anti-angiogenic effects on vascular endothelial cells, which may contribute to development of AOD in patients with CML.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/efectos adversos , Pirimidinas/efectos adversos , Enfermedades Vasculares/inducido químicamente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apolipoproteínas E/genética , Aterosclerosis/inducido químicamente , Endotelio Vascular/citología , Femenino , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana EdadRESUMEN
Proteomic-based drug testing is an emerging approach to establish the clinical value and anti-neoplastic potential of multikinase inhibitors. The multikinase inhibitor midostaurin (PKC412) is a promising new agent used to treat patients with advanced systemic mastocytosis (SM). We examined the target interaction profiles and the mast cell (MC)-targeting effects of two pharmacologically relevant midostaurin metabolites, CGP52421 and CGP62221. All three compounds, midostaurin and the two metabolites, suppressed IgE-dependent histamine secretion in basophils and MC with reasonable IC(50) values. Midostaurin and CGP62221 also produced growth inhibition and dephosphorylation of KIT in the MC leukemia cell line HMC-1.2, whereas the second metabolite, CGP52421, which accumulates in vivo, showed no substantial effects. Chemical proteomic profiling and drug competition experiments revealed that midostaurin interacts with KIT and several additional kinase targets. The key downstream regulator FES was recognized by midostaurin and CGP62221, but not by CGP52421 in MC lysates, whereas the IgE receptor downstream target SYK was recognized by both metabolites. Together, our data show that the clinically relevant midostaurin metabolite CGP52421 inhibits IgE-dependent histamine release, but is a weak inhibitor of MC proliferation, which may have clinical implications and may explain why mediator-related symptoms improve in SM patients even when disease progression occurs.
Asunto(s)
Mastocitos/efectos de los fármacos , Mastocitosis/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Estaurosporina/análogos & derivados , Adulto , Anciano , Basófilos/efectos de los fármacos , Basófilos/fisiología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Liberación de Histamina/efectos de los fármacos , Humanos , Masculino , Mastocitos/fisiología , Mastocitosis/patología , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-kit/metabolismo , Estaurosporina/farmacologíaRESUMEN
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
BACKGROUND: The multikinase inhibitor dasatinib exerts growth-inhibitory effects in patients with imatinib-resistant chronic myeloid leukaemia (CML). In first clinical trials, side effects of dasatinib, 140 mg daily, were reported to be mild and tolerable. PATIENTS AND METHODS: We examined the side effect profile in 16 patients with imatinib-resistant CML who received 140 mg dasatinib daily in our center. RESULTS: Dasatinib produced substantial and sometimes severe or even life-threatening side effects with > or = 10% body weight loss (6/16 patients), pleural effusions grade II or higher (12/16) and infectious complications (12/16), including atypical infections not seen in imatinib-treated patients. One patient developed Epstein-Barr-Virus-positive mucosal leucoplakia, one died from pneumonia caused by pneumocystis carinii and three patients developed a skin-cancer. Most events were recorded within the first 2 years of therapy, only skin tumours developed after the second year. In ex vivo experiments performed in dasatinib-treated patients, transient suppression of IgE-dependent activation of blood basophils and TcR-dependent activation of T-lymphocytes was found. Moreover, in drug-binding studies, dasatinib was found to bind to several key kinase-targets of the immune system including Lyn and Btk, in mast cell, basophil, B-cell and T-cell lines. CONCLUSION: Dasatinib acts not only anti-neoplastic in CML but may also act as an immunosuppressive agent when applied at 140 mg daily, and produces frequent pleural effusions and weight loss in advanced CML.
Asunto(s)
Antígenos de Neoplasias/inmunología , Antineoplásicos/efectos adversos , Inmunosupresores/efectos adversos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Linfocitos/inmunología , Pirimidinas/efectos adversos , Tiazoles/efectos adversos , Adulto , Anciano , Antígenos de Neoplasias/efectos de los fármacos , Antineoplásicos/administración & dosificación , Basófilos/efectos de los fármacos , Basófilos/inmunología , Dasatinib , Femenino , Citometría de Flujo , Humanos , Inmunoglobulinas/sangre , Inmunoglobulinas/efectos de los fármacos , Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Proteoma/análisis , Pirimidinas/administración & dosificación , Tiazoles/administración & dosificaciónRESUMEN
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
BACKGROUND: Urdamycin A, the principle product of Streptomyces fradiae Tü2717, is an angucycline-type antibiotic. The polyketide-derived aglycone moiety is glycosylated at two positions, but only limited information is available about glycosyltransferases involved in urdamycin biosynthesis. RESULTS: To determine the function of three glycosyltransferase genes in the urdamycin biosynthetic gene cluster, we have carried out gene inactivation and expression experiments. Inactivation of urdGT1a resulted in the predominant accumulation of urdamycin B. A mutant lacking urdGT1b and urdGT1c mainly produced compound 100-2. When urdGT1c was expressed in the urdGT1b/urdGT1c double mutant, urdamycin G and urdamycin A were detected. The mutant lacking all three genes mainly accumulated aquayamycin and urdamycinone B. Expression of urdGT1c in the triple mutant led to the formation of compound 100-1, whereas expression of urdGT1a resulted in the formation of compound 100-2. Co-expression of urdGT1b and urdGT1c resulted in the production of 12b-derhodinosyl-urdamycin A, and co-expression of urdGT1a, urdGT1b and urdGT1c resulted in the formation of urdamycin A. CONCLUSIONS: Analysis of glycosyltransferase genes of the urdamycin biosynthetic gene cluster led to an unambiguous assignment of each glycosyltransferase to a certain biosynthetic saccharide attachment step.