RESUMEN
FMS-like tyrosine kinase 3 (FLT3) in hematopoietic cells binds to its ligand at the plasma membrane (PM), then transduces growth signals. FLT3 gene alterations that lead the kinase to assume its permanently active form, such as internal tandem duplication (ITD) and D835Y substitution, are found in 30-40% of acute myelogenous leukemia (AML) patients. Thus, drugs for molecular targeting of FLT3 mutants have been developed for the treatment of AML. Several groups have reported that compared with wild-type FLT3 (FLT3-wt), FLT3 mutants are retained in organelles, resulting in low levels of PM localization of the receptor. However, the precise subcellular localization of mutant FLT3 remains unclear, and the relationship between oncogenic signaling and the mislocalization is not completely understood. In this study, we show that in cell lines established from leukemia patients, endogenous FLT3-ITD but not FLT3-wt clearly accumulates in the perinuclear region. Our co-immunofluorescence assays demonstrate that Golgi markers are co-localized with the perinuclear region, indicating that FLT3-ITD mainly localizes to the Golgi region in AML cells. FLT3-ITD biosynthetically traffics to the Golgi apparatus and remains there in a manner dependent on its tyrosine kinase activity. Tyrosine kinase inhibitors, such as quizartinib (AC220) and midostaurin (PKC412), markedly decrease FLT3-ITD retention and increase PM levels of the mutant. FLT3-ITD activates downstream in the endoplasmic reticulum (ER) and the Golgi apparatus during its biosynthetic trafficking. Results of our trafficking inhibitor treatment assays show that FLT3-ITD in the ER activates STAT5, whereas that in the Golgi can cause the activation of AKT and ERK. We provide evidence that FLT3-ITD signals from the early secretory compartments before reaching the PM in AML cells.
Asunto(s)
Proliferación Celular/genética , Leucemia Mieloide Aguda/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Mutación , Secuencias Repetidas en Tándem/genética , Tirosina Quinasa 3 Similar a fms/biosíntesis , Tirosina Quinasa 3 Similar a fms/genética , Benzotiazoles/farmacología , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Aparato de Golgi/metabolismo , Humanos , Leucemia Mieloide Aguda/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Oncogenes , Compuestos de Fenilurea/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción STAT5/metabolismo , Estaurosporina/análogos & derivados , Estaurosporina/farmacología , Células THP-1 , Proteínas Supresoras de Tumor/metabolismo , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidoresRESUMEN
BACKGROUND: KIT tyrosine kinase is expressed in mast cells, interstitial cells of Cajal, and hematopoietic cells. Permanently active KIT mutations lead these host cells to tumorigenesis, and to such diseases as mast cell leukemia (MCL), gastrointestinal stromal tumor (GIST), and acute myeloid leukemia (AML). Recently, we reported that in MCL, KIT with mutations (D816V, human; D814Y, mouse) traffics to endolysosomes (EL), where it can then initiate oncogenic signaling. On the other hand, KIT mutants including KITD814Y in GIST accumulate on the Golgi, and from there, activate downstream. KIT mutations, such as N822K, have been found in 30% of core binding factor-AML (CBF-AML) patients. However, how the mutants are tyrosine-phosphorylated and where they activate downstream molecules remain unknown. Moreover, it is unclear whether a KIT mutant other than KITD816V in MCL is able to signal on EL. METHODS: We used leukemia cell lines, such as Kasumi-1 (KITN822K, AML), SKNO-1 (KITN822K, AML), and HMC-1.1 (KITV560G, MCL), to explore how KIT transduces signals in these cells and to examine the signal platform for the mutants using immunofluorescence microscopy and inhibition of intracellular trafficking. RESULTS: In AML cell lines, KITN822K aberrantly localizes to EL. After biosynthesis, KIT traffics to the cell surface via the Golgi and immediately migrates to EL through endocytosis in a manner dependent on its kinase activity. However, results of phosphorylation imaging show that KIT is preferentially activated on the Golgi. Indeed, blockade of KITN822K migration to the Golgi with BFA/M-COPA inhibits the activation of KIT downstream molecules, such as AKT, ERK, and STAT5, indicating that KIT signaling occurs on the Golgi. Moreover, lipid rafts in the Golgi play a role in KIT signaling. Interestingly, KITV560G in HMC-1.1 migrates and activates downstream in a similar manner to KITN822K in Kasumi-1. CONCLUSIONS: In AML, KITN822K mislocalizes to EL. Our findings, however, suggest that the mutant transduces phosphorylation signals on lipid rafts of the Golgi in leukemia cells. Unexpectedly, the KITV560G signal platform in MCL is similar to that of KITN822K in AML. These observations provide new insights into the pathogenic role of KIT mutants as well as that of other mutant molecules.
Asunto(s)
Aparato de Golgi/metabolismo , Leucemia Mieloide Aguda/patología , Microdominios de Membrana/metabolismo , Mutación , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Endocitosis/genética , Activación Enzimática/genética , Humanos , Transporte de Proteínas/genéticaRESUMEN
The strain delay index is reportedly a marker of dyssynchrony and residual myocardial contractility. The aim of this study was to test the hypothesis that a relatively simple version of the strain dyssynchrony index (SDI) can predict response to cardiac resynchronization therapy (CRT) and that combining assessment of radial, circumferential, and longitudinal SDI can further improve the prediction of responders. A total of 52 patients who underwent CRT were studied. The SDI was calculated as the average difference between peak and end-systolic strain from 6 segments for radial and circumferential SDI and 18 segments for longitudinal SDI. Conventional dyssynchrony measures were assessed by interventricular mechanical delay, the Yu index, and radial dyssynchrony by speckle tracking strain. Response was defined as a ≥15% decrease in end-systolic volume after 3 months. Of the individual parameters, radial SDI ≥6.5% was the best predictor of response to CRT, with sensitivity of 81%, specificity of 81%, and an area under the curve of 0.87 (p <0.001). Circumferential SDI ≥3.2% and longitudinal SDI ≥3.6% were also found to be predictive of response to CRT, with areas under the curve of 0.81 and 0.80, respectively (p <0.001). Moreover, radial, circumferential, and longitudinal SDI at baseline were correlated with reduction of end-systolic volume with CRT. In addition, the response rate in patients with 3 positive SDIs was 100%. In contrast, rates in patients with either 1 or no positive SDIs were 42% and 22%, respectively (p <0.005 and p <0.001 vs 3 positive SDIs). In conclusion, the SDI can successfully predict response to CRT, and the combined approach leads to more accurate prediction than using individual parameters.