RESUMEN
Acute myeloid leukemia (AML) with nucleophosmin 1 (NPM1) mutations exhibits distinct biological and clinical features, accounting for approximately one-third of AML. Recently, the N 6-methyladenosine (m6A) RNA modification has emerged as a new epigenetic modification to contribute to tumorigenesis and development. However, there is limited knowledge on the role of m6A modifications in NPM1-mutated AML. In this study, the decreased m6A level was first detected and high expression of fat mass and obesity-associated protein (FTO) was responsible for the m6A suppression in NPM1-mutated AML. FTO upregulation was partially induced by NPM1 mutation type A (NPM1-mA) through impeding the proteasome pathway. Importantly, FTO promoted leukemic cell survival by facilitating cell cycle and inhibiting cell apoptosis. Mechanistic investigations demonstrated that FTO depended on its m6A RNA demethylase activity to activate PDGFRB/ERK signaling axis. Our findings indicate that FTO-mediated m6A demethylation plays an oncogenic role in NPM1-mutated AML and provide a new layer of epigenetic insight for future treatments of this distinctly leukemic entity.
RESUMEN
Acute myeloid leukaemia (AML) carrying nucleophosmin (NPM1) mutations has been defined as a distinct entity of acute leukaemia. Despite remarkable improvements in diagnosis and treatment, the long-term outcomes for this entity remain unsatisfactory. Emerging evidence suggests that leukaemia, similar to other malignant diseases, employs various mechanisms to evade killing by immune cells. However, the mechanism of immune escape in NPM1-mutated AML remains unknown. In this study, both serum and leukemic cells from patients with NPM1-mutated AML impaired the immune function of CD8+ T cells in a co-culture system. Mechanistically, leukemic cells secreted miR-19a-3p into the tumour microenvironment (TME) via small extracellular vesicles (sEVs), which was controlled by the NPM1-mutated protein/CCCTC-binding factor (CTCF)/poly (A)-binding protein cytoplasmic 1 (PABPC1) signalling axis. sEV-related miR-19a-3p was internalized by CD8+ T cells and directly repressed the expression of solute-carrier family 6 member 8 (SLC6A8; a creatine-specific transporter) to inhibit creatine import. Decreased creatine levels can reduce ATP production and impair CD8+ T cell immune function, leading to immune escape by leukemic cells. In summary, leukemic cell-derived sEV-related miR-19a-3p confers immunosuppression to CD8+ T cells by targeting SLC6A8-mediated creatine import, indicating that sEV-related miR-19a-3p might be a promising therapeutic target for NPM1-mutated AML.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Creatina/metabolismo , Vesículas Extracelulares/metabolismo , Tolerancia Inmunológica , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Mutación , Proteínas del Tejido Nervioso/metabolismo , Nucleofosmina/genética , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/metabolismo , Transducción de Señal/inmunología , Adulto , Anciano , Transporte Biológico , Técnicas de Cocultivo/métodos , Femenino , Humanos , Leucemia Mieloide Aguda/sangre , Masculino , MicroARNs/metabolismo , Persona de Mediana Edad , Escape del Tumor , Microambiente Tumoral/inmunologíaRESUMEN
BACKGROUND: Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1), which displays a distinct long noncoding RNA (lncRNA) expression profile, has been defined as a unique subgroup in the new classification of myeloid neoplasms. However, the biological roles of key lncRNAs in the development of NPM1-mutated AML are currently unclear. Here, we aimed to investigate the functional and mechanistic roles of the lncRNA HOTAIRM1 in NPM1-mutated AML. METHODS: The expression of HOTAIRM1 was analyzed with a public database and further determined by qRT-PCR in NPM1-mutated AML samples and cell lines. The cause of upregulated HOTAIRM1 expression was investigated by luciferase reporter, chromatin immunoprecipitation and ubiquitination assays. The functional role of HOTAIRM1 in autophagy and proliferation was evaluated using western blot analysis, immunofluorescence staining, a Cell Counting Kit-8 (CCK-8) assay, a 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, flow cytometric analyses and animal studies. The action mechanism of HOTAIRM1 was explored through RNA fluorescence in situ hybridization, RNA pulldown and RNA immunoprecipitation assays. RESULTS: HOTAIRM1 was highly expressed in NPM1-mutated AML. High HOTAIRM1 expression was induced in part by mutant NPM1 via KLF5-dependent transcriptional regulation. Importantly, HOTAIRM1 promoted autophagy and proliferation both in vitro and in vivo. Mechanistic investigations demonstrated that nuclear HOTAIRM1 promoted EGR1 degradation by serving as a scaffold to facilitate MDM2-EGR1 complex formation, while cytoplasmic HOTAIRM1 acted as a sponge for miR-152-3p to increase ULK3 expression. CONCLUSIONS: Taken together, our findings identify two oncogenic regulatory axes in NPM1-mutated AML centered on HOTAIRM1: one involving EGR1 and MDM2 in the nucleus and the other involving the miR-152-3p/ULK3 axis in the cytoplasm. Our study indicates that HOTAIRM1 may be a promising therapeutic target for this distinct leukemia subtype.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Regulación Neoplásica de la Expresión Génica , Leucemia Mieloide Aguda/patología , MicroARNs/genética , Mutación , Nucleofosmina/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Apoptosis , Autofagia , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Femenino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Pronóstico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Objective To investigate the effect of miR-148b-3p on the proliferation and autophagy of acute myeloid leukemia (AML) cells and its molecular mechanism. Methods Based on GEO and TCGA databases, the expression of miR-148b-3p in AML cells and its association with clinical prognosis of patients were analyzed with the bioinformatics software. The expression of miR-148b-3p in AML cells was detected by real-time quantitative PCR. The miR-148b-3p mimic and the miR-148b-3p inhibitor were transiently transfected into AML cell lines THP-1 and NB4 by liposome-mediated transfection, respectively. The proliferation of leukemia cells was evaluated by CCK-8 assay and 5-ethynyl-2'-deoxyuridine (EdU) labeling, and the protein levels of Bcl2, Bcl2-associated X protein (BAX), autophagy marker LC3, P62, and autophagy-related gene 14 (ATG14) were detected by Western blotting. The targeted binding of miR-148b-3p to ATG14 was measured by dual-luciferase reporter gene assay, and the effect of miR-148b-3p/ATG14 axis on the phenotype of AML cells was observed in the rescue experiments. Results A decreased expression of miR-148b-3p was found in leukemia blasts of AML patients, and the overall survival rate of AML patients with low expression of miR-148b-3p was significantly lower than that of the control group. Overexpression of miR-148b-3p inhibited THP-1 cells proliferation, promoted their apoptosis, downregulated the LC3II and ATG14 protein levels, and upregulated the P62 protein levels, while inhibiting the expression of miR-148b-3p in NB4 cells had the opposite effect. miR-148b-3p significantly reduced the luciferase activity of the wild-type ATG14 expression vector. The results of rescue experiments showed that overexpression of ATG14 reversed the inhibitory effect of miR-148b-3p upregulation on cell proliferation and autophagy, while inhibition of ATG14 expression weakened the promotive effect of miR-148b-3p downregulation on cell phenotype. Conclusion The miR-148b-3p inhibits the in vitro proliferation and autophagy of AML cells by targeting ATG14.
Asunto(s)
Leucemia Mieloide Aguda , MicroARNs/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Apoptosis , Autofagia/genética , Proteínas Relacionadas con la Autofagia/genética , Proliferación Celular , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , MicroARNs/genéticaRESUMEN
Nucleophosmin (NPM1) mutations are the most frequent genetic alteration in acute myeloid leukemia (AML) and aberrant cytoplasm-dislocated NPM1 mutant is a distinct biological characterization of this disease. Our group previously reported that NPM1 mutant elevated autophagy activity and autophagy activation contributed to leukemic cell survival. However, the molecular mechanisms by which cytoplasmic NPM1 mutant involving in the autophagy pathway has not been fully elucidated. Here, we showed that Unc-51-like kinase 1 (ULK1) as a core autophagy protein was highly expressed in NPM1-mA positive OCI-AML3 cells and primary NPM1-mutated AML blasts. Meanwhile, we found that NPM1-mA could interact with ULK1 protein and positively regulated ULK1 protein levels. Mechanically, NPM1-mA promoted TRAF6-dependent K63 ubiquitination and further maintained ULK1 stability and kinase activity via miR-146a. In addition, ULK1 high expression-mediated autophagy activation and facilitated to leukemic cell proliferation. Finally, we demonstrated that restoring ULK1 expression, ULK1 inhibitor SBI-0206965 treatment and using shULK1 partially rescued the effect of NPM1-mA on autophagy and cell survival. In conclusion, our findings suggest that NPM1 mutant interacts with ULK1, and thus, maintains its protein stability, which is required for NPM1 mutant-mediated autophagic cell survival. These data extend our understanding of the functions of NPM1 mutant in the regulation of autophagy activation in NPM1-mutated AML.
Asunto(s)
Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Autofagia , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Nucleares/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/antagonistas & inhibidores , Benzamidas/farmacología , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Estabilidad de Enzimas , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , MicroARNs/genética , MicroARNs/metabolismo , Mutación , Proteínas Nucleares/genética , Nucleofosmina , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , UbiquitinaciónRESUMEN
Non-small cell lung cancer is the most common type of cancer with a poor prognosis, and development of an effective diagnostic method is urgently needed. Exosomal lncRNAs, a class of transcripts longer than 200 nucleotides packaged into exosomes, have been defined as an ideal diagnostic biomarker for cancer. However, little is known about the clinical utility of exosomal lncRNAs in NSCLC. Here, we aimed to identify exosomal lncRNAs as promising biomarkers for NSCLC diagnosis. First, serum exosomes from NSCLC patients were successfully isolated by a polymer precipitation kit and then identified by TEM, NTA and western blot analysis. A total of nine candidate lncRNAs were detected by qRT-PCR in a training set. The two exosomal lncRNA TBILA and AGAP2-AS1 were screened out for the higher levels in NSCLC patients than that of healthy controls in a validation set. And there was a significant positive correlation between these exosomal lncRNAs levels and tumor size, lymph node metastasis and TNM stage. Additionally, we validated that these exosomal lncRNAs were stable in serum. Next, we evaluated the diagnostic efficiency of exosomal lncRNAs in NSCLC patients by ROC curve analysis. The data showed that individual TBILA or AGAP2-AS1 exhibited better diagnostic efficiency in NSCLC patients with different tumor pathologic subtypes and early stage, whereas the combination of lncRNAs did not provide better results than individual lncRNAs. Notably, the combination of two exosomal lncRNAs and the serum tumor biomarker Cyfra21-1 widely used in clinical practices further improved the diagnostic accuracy for NSCLC patients. This study suggests that exosomal lncRNA TBILA and AGAP2-AS1 may be promising biomarkers for diagnosis of NSCLC.
Asunto(s)
Biomarcadores de Tumor/sangre , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Exosomas/metabolismo , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/diagnóstico , ARN Largo no Codificante/metabolismo , Anciano , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Persona de Mediana Edad , ARN Largo no Codificante/sangre , ARN Largo no Codificante/genéticaRESUMEN
Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) is acknowledged as a distinct leukemia entity in the 2016 updated World Health Organization (WHO) classification. NPM1-mutated AML patients are correlated with higher extramedullary involvement. Epithelial-mesenchymal transition (EMT) is one of the key steps which cause distant metastasis in tumor. However, whether EMT-related programs contribute to cell invasion in NPM1-mutated AML remains unclear. In this study, we identified the EMT-related gene versican (VCAN) in NPM1-mutated AML across three patient datasets. Further experiments validated the elevated VCAN expression in NPM1-mutated AML primary blasts and OCI-AML3 cells with NPM1 mutation. Mechanistic studies revealed that increased VCAN expression was at least partially regulated by NPM1 mutant via TGF-ß/cPML/Smad signalling. Functional evaluations showed that silencing VCAN by shRNA significantly suppressed cell migration and invasion capacity, whereas increased VCAN by overexpressing NPM1-mA enhanced migration and invasion ability of leukemia cells. Finally, we found that high expression of VCAN was associated with poor prognosis in AML patients. These findings provide insights into the involvement of EMT-related gene VCAN in the pathogenesis of NPM1-mutated leukemia, which suggests that VCAN is an attractive target for novel diagnostic and therapeutic strategies in NPM1-mutated AML.
RESUMEN
Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) has been defined as a distinct leukemia entity in the 2016 updated WHO classification of myeloid neoplasm. Our previous report showed that autophagic activity was elevated in NPM1-mutated AML, but the underlying molecular mechanisms remain elusive. Mount of study provides evidence that glycometabolic enzymes are implicated in the autophagic process. Pyruvate kinase isoenzyme M2 (PKM2), a key glycolytic enzyme, has been recently reported as a tumor supporter in leukemia. However, little is known about the roles of PKM2 in autophagic activity in NPM1-mutated AML. In this study, PKM2 highly expressed in NPM1-mutated AML, and partially, high levels of PKM2 were upregulated by PTBP1. Further experiments demonstrated that PKM2 mediated autophagic activation and increased the phosphorylation of key autophagy protein Beclin-1. Importantly, functional experiments demonstrated that PKM2 contributed to cell survival via autophagic activation. Ultimately, high PKM2 expression was associated with short overall and event-free survival time in NPM1-mutated AML patients. Our findings indicate for the first time that glycolytic enzyme PKM2 mediates autophagic activation and further contributes to cell survival in NPM1-mutated AML, suggesting that PKM2 may serve as a promising target for treatment of NPM1-mutated AML.