RESUMO
Internal tandem duplication (ITD) mutations within FMS-like tyrosine kinase-3 (FLT3) occur in up to 30% of acute myeloid leukemia (AML) patients and confer a very poor prognosis. The oncogenic form of FLT3 is an important therapeutic target, and inhibitors specifically targeting FLT3 kinase can induce complete remission; however, relapse after remission has been observed due to acquired resistance with secondary mutations in FLT3, highlighting the need for new strategies to target FLT3-ITD mutations. Recent studies have reported that the aberrant formations of circular RNAs (circRNAs) are biological tumorigenesis-relevant mechanisms and potential therapeutic targets. Herein, we discovered a circRNA, circMYBL2, derived from the cell-cycle checkpoint gene MYBL2. circMYBL2 is more highly expressed in AML patients with FLT3-ITD mutations than in those without the FLT3-ITD mutation. We found that circMYBL2 knockdown specifically inhibits proliferation and promotes the differentiation of FLT3-ITD AML cells in vitro and in vivo. Interestingly, we found that circMYBL2 significantly influences the protein level of mutant FLT3 kinase, which contributes to the activation of FLT3-ITD-dependent signaling pathways. Mechanistically, circMYBL2 enhanced the translational efficiency of FLT3 kinase by increasing the binding of polypyrimidine tract-binding protein 1 (PTBP1) to FLT3 messenger RNA. Moreover, circMYBL2 knockdown impaired the cytoactivity of inhibitor-resistant FLT3-ITD+ cells, with a significant decrease in FLT3 kinase expression, followed by the inactivation of its downstream pathways. In summary, we are the first to reveal a circRNA that specifically influences FLT3-ITD AML and regulates FLT3 kinase levels through translational regulation, suggesting that circMYBL2 may be a potential therapeutic target for FLT3-ITD AML.
Assuntos
Proteínas de Ciclo Celular/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Leucemia Mieloide Aguda/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , RNA Circular/genética , Transativadores/genética , Tirosina Quinase 3 Semelhante a fms/genética , Animais , Linhagem Celular Tumoral , Progressão da Doença , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Biossíntese de Proteínas , Sequências de Repetição em TandemRESUMO
The long noncoding RNA ANRIL has been found to be abnormally expressed and play important roles in different cancers. However, the expression and function of ANRIL in acute myeloid leukemia (AML) remain to be declared. In this study, we found that ANRIL is up-regulated in AML patients at diagnosis and down-regulated in patients after complete remission (CR). Functional studies showed that knockdown of ANRIL expression resulted in a decline in glucose uptake and inhibition of AML cell maintenance in vitro and in vivo. Mechanically, ANRIL was found to repress the expression of Adiponectin receptor (AdipoR1), a key regulator of glucose metabolism. Both ANRIL and AdipoR1 knockdown reduced the expression levels of phosphorylation of AMPK and SIRT1, implying a previously unappreciated ANRIL-AdipoR1-AMPK/SIRT1 signaling pathway in regulating cell glucose metabolism and survival in AML. The study is the first to demonstrate that ANRIL promotes malignant cell survival and cell glucose metabolism to accelerate AML progression and is a potential prognostic marker and therapeutic target in AML treatment.
Assuntos
Glucose/metabolismo , Leucemia Mieloide Aguda/genética , Redes e Vias Metabólicas , RNA Longo não Codificante/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Adiponectina/metabolismo , Sirtuína 1/genética , Regulação para CimaRESUMO
Immunoevasion is a hallmark of cancer progression, and immune checkpoint blockade has emerged as a promising strategy for cancer treatment. microRNAs (miRNAs) are important negative regulators of gene expression in the immune system. Here, we demonstrate that miR-708 regulates CD47, a transmembrane protein that inhibits phagocytosis in T cell acute lymphoblastic leukemia. miR-708 directly targeted CD47 through binding to 3'UTR and is inversely correlated with CD47 expression. Functional studies showed that restoration of miR-708 expression in the T-ALL cell line is sufficient to promote phagocytosis by macrophages in the absence or presence of the anti-CD47 antibody to eradicate T-ALL cells, and inhibited tumor engraftment in vivo. Together, our findings suggest that miR-708 is a key negative regulator of CD47 and may serve as an attractive candidate for immunotherapy of T-ALL.
Assuntos
Antígeno CD47/genética , Regulação Leucêmica da Expressão Gênica , MicroRNAs/genética , Fagocitose/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Interferência de RNA , Linhagem Celular Tumoral , Humanos , Fagocitose/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologiaRESUMO
Small nucleolar RNAs (snoRNAs) are commonly acknowledged as a class of homogeneous non-coding RNAs that guide ribosomal RNA modifications. However, snoRNAs referred to as orphans have largely unknown functions. Here, we systematically profile chromatin-associated snoRNAs (casnoRNAs) in mammalian cells and identify a subgroup of orphan casnoRNAs responding to DNA damage stress, among which SNORA73 shows the most marked reduction in chromatin enrichment. Downregulated SNORA73 maintains cancer genome stability and differentiation block in hematopoietic malignancy. Mechanistically, casnoRNA the 5' end non-canonical structure of SNORA73 is critical for its function and binding to poly (ADP-ribose) polymerase 1 (PARP1). SNORA73 inhibits PARP1 auto-PARylation to affect cancer genome stability by forming a small nucleolar ribonucleoprotein (snoRNP) with PARP1 and canonical H/ACA proteins DKC1/NHP2. Our findings reveal the role of an orphan snoRNA serving as casnoRNA and highlights a link between non-canonical structure of snoRNA and their functional diversity.
Assuntos
Cromatina , RNA Nucleolar Pequeno , Animais , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/genética , Cromatina/genética , Dano ao DNA/genética , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genéticaRESUMO
BACKGROUND: Long noncoding enhancer RNAs (lnc-eRNAs) are a subset of stable eRNAs identified from annotated lncRNAs. They might act as enhancer activity-related therapeutic targets in cancer. However, the underlying mechanism of epigenetic activation and their function in cancer initiation and progression remain largely unknown. RESULTS: We identify a set of lncRNAs as lnc-eRNAs according to the epigenetic signatures of enhancers. We show that these lnc-eRNAs are broadly activated in MLL-rearranged leukemia (MLL leukemia), an aggressive leukemia caused by a chromosomal translocation, through a mechanism by which the HOXA cluster initiates enhancer activity, and the epigenetic reader BRD4 cooperates with the coregulator MLL fusion oncoprotein to induce transcriptional activation. To demonstrate the functional roles of lnc-eRNAs, two newly identified lnc-eRNAs transcribed from the SEELA eRNA cluster (SEELA), SEELA1 and SEELA2, are chosen for further studies. The results show that SEELA mediated cis-activated transcription of the nearby oncogene Serine incorporate 2 (SERINC2) by directly binding to the K31 amino acid (aa) of histone H4. Chromatin-bound SEELA strengthens the interaction between chromatin and histone modifiers to promote histone recognition and oncogene transcription. Further studies show that the SEELA-SERINC2 axis regulated aspects of cancer metabolism, such as sphingolipid synthesis, to affect leukemia progression. CONCLUSIONS: This study shows that lnc-eRNAs are epigenetically activated by cancer-initiating oncoproteins and uncovers a cis-activating mechanism of oncogene transcription control based on lnc-eRNA-mediated epigenetic regulation of enhancer activity, providing insights into the critical roles of lnc-eRNAs in cancer initiation and progression.
Assuntos
Histonas/genética , Histonas/metabolismo , Leucemia/genética , RNA Longo não Codificante/genética , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proliferação de Células , Elementos Facilitadores Genéticos , Epigênese Genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Proteínas de Membrana/genética , Esfingolipídeos , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Aberrant chromosomal translocations leading to tumorigenesis have been ascribed to the heterogeneously oncogenic functions. However, how fusion transcripts exporting remains to be declared. Here, we showed that the nuclear speckle-specific long noncoding RNA MALAT1 controls chimeric mRNA export processes and regulates myeloid progenitor cell differentiation in malignant hematopoiesis. We demonstrated that MALAT1 regulates chimeric mRNAs export in an m6A-dependent manner and thus controls hematopoietic cell differentiation. Specifically, reducing MALAT1 or m6A methyltransferases and the 'reader' YTHDC1 result in the universal retention of distinct oncogenic gene mRNAs in nucleus. Mechanically, MALAT1 hijacks both the chimeric mRNAs and fusion proteins in nuclear speckles during chromosomal translocations and mediates the colocalization of oncogenic fusion proteins with METTL14. MALAT1 and fusion protein complexes serve as a functional loading bridge for the interaction of chimeric mRNA and METTL14. This study demonstrated a universal mechanism of chimeric mRNA transport that involves lncRNA-fusion protein-m6A autoregulatory loop for controlling myeloid cell differentiation. Targeting the lncRNA-triggered autoregulatory loop to disrupt chimeric mRNA transport might represent a new common paradigm for treating blood malignancies.
Assuntos
Núcleo Celular/metabolismo , Leucemia/genética , RNA Longo não Codificante/metabolismo , Transporte Ativo do Núcleo Celular , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Progressão da Doença , Regulação Leucêmica da Expressão Gênica , Rearranjo Gênico/genética , Humanos , Leucemia/patologia , Masculino , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fatores de Processamento de RNA/metabolismo , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismoRESUMO
BACKGROUND: Mixed-lineage leukemia (MLL) gene rearrangements trigger aberrant epigenetic modification and gene expression in hematopoietic stem and progenitor cells, which generates one of the most aggressive subtypes of leukemia with an apex self-renewal. It remains a challenge to directly inhibit rearranged MLL itself because of its multiple fusion partners and the poorly annotated downstream genes of MLL fusion proteins; therefore, novel therapeutic targets are urgently needed. METHODS: qRT-PCR, receiver operating characteristic (ROC), and leukemia-free survival analysis were used to validate LAMP5-AS1 (LAMP5 antisense 1) expression and evaluate its clinical value. We performed in vitro and in vivo experiments to investigate the functional relevance of LAMP5-AS1 in MLL leukemia progression and leukemia cell stemness. RNA electrophoretic mobility shift assays (EMSA), histone methyltransferase assay, RNA pull-down assay, and RNA fluorescence in situ hybridization (FISH) were used to validate the relationship between LAMP5-AS1 and the methyltransferase activity of DOT1L. The downstream ectopic target genes of LAMP5-AS1/DOT1L were validated by the chromatin immunoprecipitation (ChIP) and western blot. RESULTS: We discovered that a long noncoding RNA (lncRNA) LAMP5-AS1 can promote higher degrees of H3K79 methylation, followed by upregulated expression of the self-renewal genes in the HOXA cluster, which are responsible for leukemia stemness in context of MLL rearrangements. We found that LAMP5-AS1 is specifically overexpressed in MLL leukemia patients (n = 58) than that in the MLL-wt leukemia (n = 163) (p < 0.001), and the patients with a higher expression level of LAMP5-AS1 exhibited a reduced 5-year leukemia-free survival (p < 0.01). LAMP5-AS1 suppression significantly reduced colony formation and increased differentiation of primary MLL leukemia CD34+ cells. Mechanistically, LAMP5-AS1 facilitated the methyltransferase activity of DOT1L by directly binding its Lys-rich region of catalytic domain, thus promoting the global patterns of H3K79 dimethylation and trimethylation in cells. These observations supported that LAMP5-AS1 upregulated H3K79me2/me3 and the transcription of DOT1L ectopic target genes. CONCLUSIONS: This is the first study that a lncRNA regulates the self-renewal program and differentiation block in MLL leukemia cells by facilitating the methyltransferase activity of DOT1L and global H3K79 methylation, showing its potential as a therapeutic target for MLL leukemia.
Assuntos
Autorrenovação Celular/genética , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Membrana Lisossomal/genética , Células-Tronco Neoplásicas/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , RNA Antissenso/genética , RNA Neoplásico/genética , Animais , Pré-Escolar , Feminino , Regulação Leucêmica da Expressão Gênica/genética , Vetores Genéticos/genética , Xenoenxertos , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Lactente , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Lisina/metabolismo , Masculino , Metilação , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Cultura Primária de Células , Processamento de Proteína Pós-Traducional , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Organismos Livres de Patógenos Específicos , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
Chromosomal translocation leads to the juxtaposition of two otherwise separate DNA loci, which could result in gene fusion. These rearrangements at the DNA level are catastrophic events and often have causal roles in tumorigenesis. The oncogenic DNA messages are transferred to RNA molecules, which are in most cases translated into cancerous fusion proteins. Gene expression programs and signaling pathways are altered in these cytogenetically abnormal contexts. Notably, non-coding RNAs have attracted increasing attention and are believed to be tightly associated with chromosome-rearranged cancers. These RNAs not only function as modulators in downstream pathways but also directly affect chromosomal translocation or the associated products. This review summarizes recent research advances on the relationship between non-coding RNAs and chromosomal translocations and on diverse functions of non-coding RNAs in cancers with chromosomal rearrangements.
Assuntos
Neoplasias/genética , RNA não Traduzido/metabolismo , Translocação Genética/fisiologia , Animais , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , RNA não Traduzido/genética , Translocação Genética/genéticaRESUMO
PURPOSE: Despite many attempts to understand mixed-lineage leukemia (MLL leukemia), effective therapies for this disease remain limited. We identified a lysosome-associated membrane protein (LAMP) family member, LAMP5, that is specifically and highly expressed in patients with MLL leukemia. The purpose of the study was to demonstrate the functional relevance and clinical value of LAMP5 in the disease. EXPERIMENTAL DESIGN: We first recruited a large cohort of leukemia patients to validate LAMP5 expression and evaluate its clinical value. We then performed in vitro and in vivo experiments to investigate the functional relevance of LAMP5 in MLL leukemia progression or maintenance. RESULTS: LAMP5 was validated as being specifically and highly expressed in patients with MLL leukemia and was associated with a poor outcome. Functional studies showed that LAMP5 is a novel autophagic suppressor and protects MLL fusion proteins from autophagic degradation. Specifically targeting LAMP5 significantly promoted degradation of MLL fusion proteins and inhibited MLL leukemia progression in both an animal model and primary cells. We further revealed that LAMP5 is a direct target of the H3K79 histone methyltransferase DOT1L. Downregulating LAMP5 with a DOT1L inhibitor enhanced the selective autophagic degradation of MLL oncoproteins and extended survival in vivo; this observation was especially significant when combining DOT1L inhibitors with LAMP5 knockdown. CONCLUSIONS: This study demonstrates that LAMP5 serves as a "bodyguard" for MLL fusions to evade degradation and is the first to link H3K79 methylation to autophagy regulation, highlighting the potential of LAMP5 as a therapeutic target for MLL leukemia.
Assuntos
Biomarcadores Tumorais/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Leucemia/patologia , Proteínas de Membrana Lisossomal/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Animais , Apoptose , Autofagia , Biomarcadores Tumorais/genética , Proliferação de Células , Seguimentos , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Humanos , Leucemia/genética , Leucemia/metabolismo , Proteínas de Membrana Lisossomal/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/genética , Prognóstico , Proteólise , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Circular RNAs (circRNAs) represent a type of endogenous noncoding RNAs that are generated by back-splicing events and favor repetitive sequences. Recent studies have reported that cancer-associated chromosomal translocations could juxtapose distant complementary repetitive intronic sequences, resulting in the aberrant formation of circRNAs. However, among the reported fusion genes, only a small number of circRNAs were found to originate from fusion regions during gene translocation. We question if circRNAs could also originate from fusion partners during gene translocation. METHODS: Firstly, we designed divergent primers for qRT-PCR to identify a circRNA circAF4 in AF4 gene and investigated the expression pattern in different types of leukemia samples. Secondly, we designed two small interfering RNAs specially targeting the back-spliced junction point of circAF4 for functional studies. CCK8 cell proliferation and cell cycle assay were performed, and a NOD-SCID mouse model was used to investigate the contribution of circAF4 in leukemogenesis. Finally, luciferase reporter assay, AGO2 RNA immunoprecipitation (RIP), and RNA Fluorescent in Situ Hybridization (FISH) were performed to confirm the relationship of miR-128-3p, circAF4, and MLL-AF4 expression. RESULTS: We discovered a circRNA, named circAF4, originating from the AF4 gene, a partner of the MLL fusion gene in MLL-AF4 leukemia. We showed that circAF4 plays an oncogenic role in MLL-AF4 leukemia and promotes leukemogenesis in vitro and in vivo. More importantly, knockdown of circAF4 increases the leukemic cell apoptosis rate in MLL-AF4 leukemia cells, while no effect was observed in leukemia cells that do not carry the MLL-AF4 translocation. Mechanically, circAF4 can act as a miR-128-3p sponge, thereby releasing its inhibition on MLL-AF4 expression. We finally analyzed most of the MLL fusion genes loci and found that a number of circRNAs could originate from these partners, suggesting the potential roles of fusion gene partner-originating circRNAs (named as FP-circRNAs) in leukemia with chromosomal translocations. CONCLUSION: Our findings demonstrate that the abnormal elevated expression of circAF4 regulates the cell growth via the circAF4/miR-128-3p/MLL-AF4 axis, which could contribute to leukemogenesis, suggesting that circAF4 may be a novel therapeutic target of MLL-AF4 leukemia.