RESUMEN
RNA constitutes a large fraction of chromatin. Spatial distribution and functional relevance of most of RNA-chromatin interactions remain unknown. We established a landscape analysis of RNA-chromatin interactions in human acute myeloid leukemia (AML). In total more than 50 million interactions were captured in an AML cell line. Protein-coding mRNAs and long non-coding RNAs exhibited a substantial number of interactions with chromatin in cis suggesting transcriptional activity. In contrast, small nucleolar RNAs (snoRNAs) and small nuclear RNAs (snRNAs) associated with chromatin predominantly in trans suggesting chromatin specific functions. Of note, snoRNA-chromatin interaction was associated with chromatin modifications and occurred independently of the classical snoRNA-RNP complex. Two C/D box snoRNAs, namely SNORD118 and SNORD3A, displayed high frequency of trans-association with chromatin. The transcription of SNORD118 and SNORD3A was increased upon leukemia transformation and enriched in leukemia stem cells, but decreased during myeloid differentiation. Suppression of SNORD118 and SNORD3A impaired leukemia cell proliferation and colony forming capacity in AML cell lines and primary patient samples. Notably, this effect was leukemia specific with less impact on healthy CD34+ hematopoietic stem and progenitor cells. These findings highlight the functional importance of chromatin-associated RNAs overall and in particular of SNORD118 and SNORD3A in maintaining leukemia propagation.
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Cromatina , Leucemia Mieloide Aguda , Humanos , Cromatina/metabolismo , Cromatina/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/metabolismo , ARN Pequeño no Traducido/genética , Proliferación Celular/genética , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo , Línea Celular TumoralRESUMEN
Venetoclax/azacitidine combination therapy is effective in acute myeloid leukemia (AML) and tolerable for older, multimorbid patients. Despite promising response rates, many patients do not achieve sustained remission or are upfront refractory. Identification of resistance mechanisms and additional therapeutic targets represent unmet clinical needs. By using a genome-wide CRISPR/Cas9 library screen targeting 18,053 protein- coding genes in a human AML cell line, various genes conferring resistance to combined venetoclax/azacitidine treatment were identified. The ribosomal protein S6 kinase A1 (RPS6KA1) was among the most significantly depleted sgRNA-genes in venetoclax/azacitidine- treated AML cells. Addition of the RPS6KA1 inhibitor BI-D1870 to venetoclax/azacitidine decreased proliferation and colony forming potential compared to venetoclax/azacitidine alone. Furthermore, BI-D1870 was able to completely restore the sensitivity of OCI-AML2 cells with acquired resistance to venetoclax/azacitidine. Analysis of cell surface markers revealed that RPS6KA1 inhibition efficiently targeted monocytic blast subclones as a potential source of relapse upon venetoclax/azacitidine treatment. Taken together, our results suggest RPS6KA1 as mediator of resistance towards venetoclax/azacitidine and additional RPS6KA1 inhibition as strategy to prevent or overcome resistance.
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Azacitidina , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Azacitidina/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Proteínas Quinasas S6 Ribosómicas , Proteínas Quinasas S6 Ribosómicas 90-kDa , ARN Guía de Sistemas CRISPR-CasRESUMEN
The development and regulation of malignant self-renewal remain unresolved issues. Here, we provide biochemical, genetic, and functional evidence that dynamics in ribosomal RNA (rRNA) 2'-O-methylation regulate leukemia stem cell (LSC) activity in vivo. A comprehensive analysis of the rRNA 2'-O-methylation landscape of 94 patients with acute myeloid leukemia (AML) revealed dynamic 2'-O-methylation specifically at exterior sites of ribosomes. The rRNA 2'-O-methylation pattern is closely associated with AML development stage and LSC gene expression signature. Forced expression of the 2'-O-methyltransferase fibrillarin (FBL) induced an AML stem cell phenotype and enabled engraftment of non-LSC leukemia cells in NSG mice. Enhanced 2'-O-methylation redirected the ribosome translation program toward amino acid transporter mRNAs enriched in optimal codons and subsequently increased intracellular amino acid levels. Methylation at the single site 18S-guanosine 1447 was instrumental for LSC activity. Collectively, our work demonstrates that dynamic 2'-O-methylation at specific sites on rRNAs shifts translational preferences and controls AML LSC self-renewal. SIGNIFICANCE: We establish the complete rRNA 2'-O-methylation landscape in human AML. Plasticity of rRNA 2'-O-methylation shifts protein translation toward an LSC phenotype. This dynamic process constitutes a novel concept of how cancers reprogram cell fate and function. This article is highlighted in the In This Issue feature, p. 247.
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Leucemia Mieloide Aguda , ARN Ribosómico , Humanos , Animales , Ratones , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Leucemia Mieloide Aguda/patología , Ribosomas/genética , Ribosomas/metabolismo , Metilación , Fenotipo , Células Madre Neoplásicas/metabolismoRESUMEN
FLT3 tyrosine kinase inhibitor (TKI) therapy evolved into a standard therapy in FLT3-mutated AML. TKI resistance, however, develops frequently with poor outcomes. We analyzed acquired TKI resistance in AML cell lines by multilayered proteome analyses. Leupaxin (LPXN), a regulator of cell migration and adhesion, was induced during early resistance development, alongside the tyrosine kinase PTK2B which phosphorylated LPXN. Resistant cells differed in cell adhesion and migration, indicating altered niche interactions. PTK2B and LPXN were highly expressed in leukemic stem cells in FLT3-ITD patients. PTK2B/FAK inhibition abrogated resistance-associated phenotypes, such as enhanced cell migration. Altered pathways in resistant cells, assessed by nascent proteomics, were largely reverted upon PTK2B/FAK inhibition. PTK2B/FAK inhibitors PF-431396 and defactinib synergized with different TKIs or daunorubicin in FLT3-mutated AML. Midostaurin-resistant and AML cells co-cultured with mesenchymal stroma cells responded particularly well to PTK2B/FAK inhibitor addition. Xenograft mouse models showed significant longer time to leukemia symptom-related endpoint upon gilteritinib/defactinib combination treatment in comparison to treatment with either drug alone. Our data suggest that the leupaxin-PTK2B axis plays an important role in acquired TKI resistance in AML. PTK2B/FAK inhibitors act synergistically with currently used therapeutics and may overcome emerging TKI resistance in FLT3-mutated AML at an early timepoint.
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Leucemia Mieloide Aguda , Inhibidores de Proteínas Quinasas , Animales , Benzamidas , Línea Celular Tumoral , Daunorrubicina/uso terapéutico , Resistencia a Antineoplásicos , Quinasa 2 de Adhesión Focal/genética , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Ratones , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/genética , Proteoma/genética , Pirazinas , Sulfonamidas , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/uso terapéuticoRESUMEN
BCL-2 inhibition has been shown to be effective in acute myeloid leukemia (AML) in combination with hypomethylating agents or low-dose cytarabine. However, resistance and relapse represent major clinical challenges. Therefore, there is an unmet need to overcome resistance to current venetoclax-based strategies. We performed high-throughput drug screening to identify effective combination partners for venetoclax in AML. Overall, 64 antileukemic drugs were screened in 31 primary high-risk AML samples with or without venetoclax. Gilteritinib exhibited the highest synergy with venetoclax in FLT3 wild-type AML. The combination of gilteritinib and venetoclax increased apoptosis, reduced viability, and was active in venetoclax-azacitidine-resistant cell lines and primary patient samples. Proteomics revealed increased FLT3 wild-type signaling in specimens with low in vitro response to the currently used venetoclax-azacitidine combination. Mechanistically, venetoclax with gilteritinib decreased phosphorylation of ERK and GSK3B via combined AXL and FLT3 inhibition with subsequent suppression of the antiapoptotic protein MCL-1. MCL-1 downregulation was associated with increased MCL-1 phosphorylation of serine 159, decreased phosphorylation of threonine 161, and proteasomal degradation. Gilteritinib and venetoclax were active in an FLT3 wild-type AML patient-derived xenograft model with TP53 mutation and reduced leukemic burden in 4 patients with FLT3 wild-type AML receiving venetoclax-gilteritinib off label after developing refractory disease under venetoclax-azacitidine. In summary, our results suggest that combined inhibition of FLT3/AXL potentiates venetoclax response in FLT3 wild-type AML by inducing MCL-1 degradation. Therefore, the venetoclax-gilteritinib combination merits testing as a potentially active regimen in patients with high-risk FLT3 wild-type AML.
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Leucemia Mieloide Aguda , Humanos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Azacitidina , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
Immunotherapies, such as chimeric antigen receptor (CAR) modified T cells and antibody-drug conjugates (ADCs), have revolutionized the treatment of cancer, especially of lymphoid malignancies. The application of targeted immunotherapy to patients with acute myeloid leukemia (AML) has been limited in particular by the lack of a tumor-specific target antigen. Gemtuzumab ozogamicin (GO), an ADC targeting CD33, is the only approved immunotherapeutic agent in AML. In our study, we introduce a CD33-directed third-generation CAR T-cell product (3G.CAR33-T) for the treatment of patients with AML. 3G.CAR33-T cells could be expanded up to the end-of-culture, that is, 17 days after transduction, and displayed significant cytokine secretion and robust cytotoxic activity when incubated with CD33-positive cells including cell lines, drug-resistant cells, primary blasts as well as normal hematopoietic stem and progenitor cells (HSPCs). When compared to second-generation CAR33-T cells, 3G.CAR33-T cells exhibited higher viability, increased proliferation and stronger cytotoxicity. Also, GO exerted strong antileukemia activity against CD33-positive AML cells. Upon genomic deletion of CD33 in HSPCs, 3G.CAR33-T cells and GO preferentially killed wildtype leukemia cells, while sparing CD33-deficient HSPCs. Our data provide evidence for the applicability of CD33-targeted immunotherapies in AML and its potential implementation in CD33 genome-edited stem cell transplantation approaches.
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Gemtuzumab/uso terapéutico , Trasplante de Células Madre Hematopoyéticas , Inmunoterapia Adoptiva , Leucemia Mieloide Aguda/terapia , Receptores Quiméricos de Antígenos/inmunología , Lectina 3 Similar a Ig de Unión al Ácido Siálico/inmunología , Edición Génica , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/patología , Lectina 3 Similar a Ig de Unión al Ácido Siálico/análisis , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genéticaRESUMEN
Treatment of acute myeloid leukemia (AML) remains challenging and novel targets and synergistic therapies still need to be discovered. We performed a high-throughput RNAi screen in three different AML cell lines and primary human leukemic blasts to identify genes that synergize with common antileukemic therapies. We used a pooled shRNA library that covered 5043 different genes and combined transfection with exposure to either azacytidine or cytarabine analog to the concept of synthetic lethality. Suppression of the chemokine CXCL12 ranked highly among the candidates of the cytarabine group. Azacytidine in combination with suppression of genes within the neddylation pathway led to synergistic results. NEDD8 and RBX1 inhibition by the small molecule inhibitor pevonedistat inhibited leukemia cell growth. These findings establish an in vitro synergism between NEDD8 inhibition and azacytidine in AML. Taken together, neddylation constitutes a suitable target pathway for azacytidine combination strategies.
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Azacitidina/uso terapéutico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Proteína NEDD8/metabolismo , Interferencia de ARN , Transducción de Señal/genética , Transducción de Señal/fisiología , Quimiocina CXCL12/metabolismo , Terapia Combinada , Ciclopentanos/farmacología , Ciclopentanos/uso terapéutico , Células HL-60 , Humanos , Leucemia Mieloide Aguda/metabolismo , Factor de Transcripción 1 de la Leucemia de Células Pre-B/metabolismo , Pirimidinas/farmacología , Pirimidinas/uso terapéuticoRESUMEN
Modifications of RNA commonly occur in all species. Multiple enzymes are involved as writers, erasers and readers of these modifications. Many RNA modifications or the respective enzymes are associated with human disease and especially cancer. Currently, the mechanisms how RNA modifications impact on a large number of intracellular processes are emerging and knowledge about the pathogenetic role of RNA modifications increases. In Acute Myeloid Leukemia (AML), the N6-methyladenosine (m6A) modification has emerged as an important modulator of leukemogenesis. The writer proteins METTL3 and METTL14 are both involved in AML pathogenesis and might be suitable therapeutic targets. Recently, close links between 2'-O-methylation (2'-O-me) of ribosomal RNA and leukemogenesis were discovered. The AML1-ETO oncofusion protein which specifically occurs in a subset of AML was found to depend on induction of snoRNAs and 2'-O-me for leukemogenesis. Also, NPM1, an important tumor suppressor in AML, was associated with altered snoRNAs and 2'-O-me. These findings point toward novel pathogenetic mechanisms and potential therapeutic interventions. The current knowledge and the implications are the topic of this review.
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Leucemia Mieloide Aguda , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Humanos , MetilaciónRESUMEN
Somatic mutations in DNA methyltransferase 3A (DNMT3A) are among the most frequent alterations in clonal hematopoiesis (CH) and acute myeloid leukemia (AML), with a hotspot in exon 23 at arginine 882 (DNMT3AR882). Here, we demonstrate that DNMT3AR882H-dependent CH and AML cells are specifically susceptible to the hypomethylating agent azacytidine (AZA). Addition of AZA to chemotherapy prolonged AML survival solely in individuals with DNMT3AR882 mutations, suggesting its potential as a predictive marker for AZA response. AML and CH mouse models confirmed AZA susceptibility specifically in DNMT3AR882H-expressing cells. Hematopoietic stem cells (HSCs) and progenitor cells expressing DNMT3AR882H exhibited cell autonomous viral mimicry response as a result of focal DNA hypomethylation at retrotransposon sequences. Administration of AZA boosted hypomethylation of retrotransposons specifically in DNMT3AR882H-expressing cells and maintained elevated levels of canonical interferon-stimulated genes (ISGs), thus leading to suppressed protein translation and increased apoptosis.
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ADN (Citosina-5-)-Metiltransferasas , Leucemia Mieloide Aguda , Animales , Azacitidina/farmacología , Hematopoyesis Clonal , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Metiltransferasa 3A , Células Madre Hematopoyéticas/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Ratones , MutaciónRESUMEN
All-trans-retinoic acid (ATRA) is highly active in acute promyelocytic leukemia but not in other types of acute myeloid leukemia (AML). Previously, we showed that ATRA in combination with Lysine-specific demethylase 1 (LSD1) inhibition by tranylcypromine (TCP) can induce myeloid differentiation in AML blasts. This phase I/II clinical trial investigated the safety and efficacy of TCP/ATRA treatment as salvage therapy for relapsed/refractory (r/r) AML. The combination was evaluated in 18 patients, ineligible for intensive treatment. The overall response rate was 20%, including two complete remissions without hematological recovery and one partial response. We also observed myeloid differentiation upon TCP/ATRA treatment in patients who did not reach clinical remission. Median overall survival (OS) was 3.3 months, and one-year OS 22%. One patient developed an ATRA-induced differentiation syndrome. The most frequently reported adverse events were vertigo and hypotension. TCP plasma levels correlated with intracellular TCP concentration. Increased H3K4me1 and H3k4me2 levels were observed in AML blasts and white blood cells from some TCP/ATRA treated patients. Combined TCP/ATRA treatment can induce differentiation of AML blasts and lead to clinical response in heavily pretreated patients with r/r AML with acceptable toxicity. These findings emphasize the potential of LSD1 inhibition combined with ATRA for AML treatment.
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Resistencia a Antineoplásicos/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Prueba de Estudio Conceptual , Terapia Recuperativa , Tranilcipromina/uso terapéutico , Tretinoina/uso terapéutico , Adulto , Anciano , Antidepresivos/uso terapéutico , Antineoplásicos/uso terapéutico , Proteínas de Arabidopsis , Proteínas de Unión al ADN , Quimioterapia Combinada , Femenino , Estudios de Seguimiento , Humanos , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/patología , Pronóstico , Estudios Prospectivos , Tasa de Supervivencia , Factores de Transcripción , Adulto JovenRESUMEN
Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide underlining the urgent need for new biomarkers and therapeutic targets for this disease. Long noncoding RNAs are critical players in NSCLC but the role of small RNA species is not well understood. In the present study, we investigated the role of H/ACA box small nucleolar RNAs (snoRNAs) and snoRNA-bound ribonucleoproteins (snoRNPs) in the tumorigenesis of NSCLC. H/ACA box snoRNPs including the NOP10 core protein were highly expressed in NSCLC. High levels of either NOP10 mRNA or protein were associated with poor prognosis in NSCLC patients. Loss of NOP10 and subsequent reduction of H/ACA box snoRNAs and rRNA pseudouridylation inhibited lung cancer cell growth, colony formation, migration, and invasion. A focused CRISPR/Cas9 snoRNA knockout screen revealed that genomic deletion of SNORA65, SNORA7A, and SNORA7B reduced proliferation of lung cancer cells. In line, high levels of SNORA65, SNORA7A, and SNORA7B were observed in primary lung cancer specimens with associated changes in rRNA pseudouridylation. Knockdown of either SNORA65 or SNORA7A/B inhibited growth and colony formation of NSCLC cell lines. Our data indicate that specific H/ACA box snoRNAs and snoRNA-associated proteins such as NOP10 have an oncogenic role in NSCLC providing new potential biomarkers and therapeutic targets for the disease.
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Neoplasias Pulmonares/genética , ARN Nucleolar Pequeño/genética , Ribonucleoproteínas Nucleolares Pequeñas/genética , Línea Celular Tumoral , Movimiento Celular/genética , Nucléolo Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/patología , Pronóstico , Procesamiento Postranscripcional del ARN/genética , ARN Mensajero/genéticaRESUMEN
Recent efforts reclassified B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) into more refined subtypes. Nevertheless, outcomes of relapsed BCP-ALL remain unsatisfactory, particularly in adult patients where the molecular basis of relapse is still poorly understood. To elucidate the evolution of relapse in BCP-ALL, we established a comprehensive multi-omics dataset including DNA-sequencing, RNA-sequencing, DNA methylation array and proteome MASS-spec data from matched diagnosis and relapse samples of BCP-ALL patients (n = 50) including the subtypes DUX4, Ph-like and two aneuploid subtypes. Relapse-specific alterations were enriched for chromatin modifiers, nucleotide and steroid metabolism including the novel candidates FPGS, AGBL and ZNF483. The proteome expression analysis unraveled deregulation of metabolic pathways at relapse including the key proteins G6PD, TKT, GPI and PGD. Moreover, we identified a novel relapse-specific gene signature specific for DUX4 BCP-ALL patients highlighting chemotaxis and cytokine environment as a possible driver event at relapse. This study presents novel insights at distinct molecular levels of relapsed BCP-ALL based on a comprehensive multi-omics integrated data set including a valuable proteomics data set. The relapse specific aberrations reveal metabolic signatures on genomic and proteomic levels in BCP-ALL relapse. Furthermore, the chemokine expression signature in DUX4 relapse underscores the distinct status of DUX4-fusion BCP-ALL.
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Citocinas , Regulación Leucémica de la Expresión Génica , Proteínas de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Adolescente , Adulto , Niño , Citocinas/genética , Citocinas/metabolismo , Femenino , Genómica , Humanos , Masculino , Redes y Vías Metabólicas , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/clasificación , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , ProteómicaRESUMEN
BACKGROUND: Long non-coding RNAs (lncRNAs) have emerged as a novel class of RNA due to its diverse mechanism in cancer development and progression. However, the role and expression pattern of lncRNAs in molecular subtypes of B cell acute lymphoblastic leukemia (BCP-ALL) have not yet been investigated. Here, we assess to what extent lncRNA expression and DNA methylation is driving the progression of relapsed BCP-ALL subtypes and we determine if the expression and DNA methylation profile of lncRNAs correlates with established BCP-ALL subtypes. METHODS: We performed RNA sequencing and DNA methylation (Illumina Infinium microarray) of 40 diagnosis and 42 relapse samples from 45 BCP-ALL patients in a German cohort and quantified lncRNA expression. Unsupervised clustering was applied to ascertain and confirm that the lncRNA-based classification of the BCP-ALL molecular subtypes is present in both our cohort and an independent validation cohort of 47 patients. A differential expression and differential methylation analysis was applied to determine the subtype-specific, relapse-specific, and differentially methylated lncRNAs. Potential functions of subtype-specific lncRNAs were determined by using co-expression-based analysis on nearby (cis) and distally (trans) located protein-coding genes. RESULTS: Using an integrative Bioinformatics analysis, we developed a comprehensive catalog of 1235 aberrantly dysregulated BCP-ALL subtype-specific and 942 relapse-specific lncRNAs and the methylation profile of three subtypes of BCP-ALL. The 1235 subtype-specific lncRNA signature represented a similar classification of the molecular subtypes of BCP-ALL in the independent validation cohort. We identified a strong correlation between the DUX4-specific lncRNAs and genes involved in the activation of TGF-ß and Hippo signaling pathways. Similarly, Ph-like-specific lncRNAs were correlated with genes involved in the activation of PI3K-AKT, mTOR, and JAK-STAT signaling pathways. Interestingly, the relapse-specific lncRNAs correlated with the activation of metabolic and signaling pathways. Finally, we found 23 promoter methylated lncRNAs epigenetically facilitating their expression levels. CONCLUSION: Here, we describe a set of subtype-specific and relapse-specific lncRNAs from three major BCP-ALL subtypes and define their potential functions and epigenetic regulation. The subtype-specific lncRNAs are reproducible and can effectively stratify BCP-ALL subtypes. Our data uncover the diverse mechanism of action of lncRNAs in BCP-ALL subtypes defining which lncRNAs are involved in the pathogenesis of disease and are relevant for the stratification of BCP-ALL subtypes.
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Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , ARN Largo no Codificante/genética , Transcriptoma/genética , Adulto , Secuencia de Bases/genética , Berlin , Biomarcadores de Tumor/genética , Médula Ósea , Niño , Estudios de Cohortes , Metilación de ADN/genética , Epigénesis Genética , Femenino , Humanos , Masculino , Redes y Vías Metabólicas/genética , Regiones Promotoras Genéticas/genética , RecurrenciaRESUMEN
Motivation: The application of next-generation sequencing in research and particularly in clinical routine requires valid variant calling results. However, evaluation of several commonly used tools has pointed out that not a single tool meets this requirement. False positive as well as false negative calls necessitate additional experiments and extensive manual work. Intelligent combination and output filtration of different tools could significantly improve the current situation. Results: We developed appreci8, an automatic variant calling pipeline for calling single nucleotide variants and short indels by combining and filtering the output of eight open-source variant calling tools, based on a novel artifact- and polymorphism score. Appreci8 was trained on two data sets from patients with myelodysplastic syndrome, covering 165 Illumina samples. Subsequently, appreci8's performance was tested on five independent data sets, covering 513 samples. Variation in sequencing platform, target region and disease entity was considered. All calls were validated by re-sequencing on the same platform, a different platform or expert-based review. Sensitivity of appreci8 ranged between 0.93 and 1.00, while positive predictive value ranged between 0.65 and 1.00. In all cases, appreci8 showed superior performance compared to any evaluated alternative approach. Availability and implementation: Appreci8 is freely available at https://hub.docker.com/r/wwuimi/appreci8/. Sequencing data (BAM files) of the 678 patients analyzed with appreci8 have been deposited into the NCBI Sequence Read Archive (BioProjectID: 388411; https://www.ncbi.nlm.nih.gov/bioproject/PRJNA388411). Supplementary information: Supplementary data are available at Bioinformatics online.
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Secuenciación de Nucleótidos de Alto Rendimiento , Polimorfismo de Nucleótido Simple , Programas Informáticos , Biología Computacional , Humanos , Síndromes Mielodisplásicos/genéticaRESUMEN
Leukaemogenesis requires enhanced self-renewal, which is induced by oncogenes. The underlying molecular mechanisms remain incompletely understood. Here, we identified C/D box snoRNAs and rRNA 2'-O-methylation as critical determinants of leukaemic stem cell activity. Leukaemogenesis by AML1-ETO required expression of the groucho-related amino-terminal enhancer of split (AES). AES functioned by inducing snoRNA/RNP formation via interaction with the RNA helicase DDX21. Similarly, global loss of C/D box snoRNAs with concomitant loss of rRNA 2'-O-methylation resulted in decreased leukaemia self-renewal potential. Genomic deletion of either C/D box snoRNA SNORD14D or SNORD35A suppressed clonogenic potential of leukaemia cells in vitro and delayed leukaemogenesis in vivo. We further showed that AML1-ETO9a, MYC and MLL-AF9 all enhanced snoRNA formation. Expression levels of C/D box snoRNAs in AML patients correlated closely with in vivo frequency of leukaemic stem cells. Collectively, these findings indicate that induction of C/D box snoRNA/RNP function constitutes an important pathway in leukaemogenesis.
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Proliferación Celular , Autorrenovación de las Células , Transformación Celular Neoplásica/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Leucemia/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , ARN Nucleolar Pequeño/metabolismo , Ribonucleoproteínas/metabolismo , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Proteínas Co-Represoras , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Regulación Leucémica de la Expresión Génica , Predisposición Genética a la Enfermedad , Células HEK293 , Células HL-60 , Humanos , Células K562 , Leucemia/genética , Leucemia/patología , Metilación , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Fenotipo , Mapas de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN Nucleolar Pequeño/genética , Proteína 1 Compañera de Translocación de RUNX1 , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Ribonucleoproteínas/genética , Transducción de Señal , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células U937RESUMEN
The polycomb protein enhancer of zeste homolog 2 (EZH2) may have a dual role in cancer pathogenesis acting as an oncogene or as a tumor suppressor depending on the cancer type. We recently demonstrated that proteasomal degradation of EZH2 resulting from cyclin-dependent kinase 1 (CDK1)-induced phosphorylation at Threonine (T) 487 represents a novel mechanism of drug resistance in acute myeloid leukemia (AML). Our findings suggest that restoration of EZH2 protein is a viable approach to overcome therapy resistance in AML.
RESUMEN
The transcription factor Meis1 drives myeloid leukemogenesis in the context of Hox gene overexpression but is currently considered undruggable. We therefore investigated whether myeloid progenitor cells transformed by Hoxa9 and Meis1 become addicted to targetable signaling pathways. A comprehensive (phospho)proteomic analysis revealed that Meis1 increased Syk protein expression and activity. Syk upregulation occurs through a Meis1-dependent feedback loop. By dissecting this loop, we show that Syk is a direct target of miR-146a, whose expression is indirectly regulated by Meis1 through the transcription factor PU.1. In the context of Hoxa9 overexpression, Syk signaling induces Meis1, recapitulating several leukemogenic features of Hoxa9/Meis1-driven leukemia. Finally, Syk inhibition disrupts the identified regulatory loop, prolonging survival of mice with Hoxa9/Meis1-driven leukemia.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Leucemia Mieloide Aguda/metabolismo , MicroARNs/genética , Proteínas de Neoplasias/metabolismo , Quinasa Syk/metabolismo , Animales , Regulación Leucémica de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Integrina beta3/metabolismo , Estimación de Kaplan-Meier , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Ratones Endogámicos C57BL , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Proteínas de Neoplasias/genética , Transducción de Señal , Quinasa Syk/genéticaRESUMEN
Ewing sarcomas (ES) are highly malignant, osteolytic bone or soft tissue tumors, which are characterized by EWS-ETS translocations and early metastasis to lung and bone. In this study, we investigated the role of the BRICHOS chaperone domain-containing endochondral bone protein chondromodulin I (CHM1) in ES pathogenesis. CHM1 is significantly overexpressed in ES, and chromosome immunoprecipitation (ChIP) data demonstrate CHM1 to be directly bound by an EWS-ETS translocation, EWS-FLI1. Using RNA interference, we observed that CHM1 promoted chondrogenic differentiation capacity of ES cells but decreased the expression of osteolytic genes such as HIF1A, IL6, JAG1, and VEGF. This was in line with the induction of the number of tartrate-resistant acid phosphatase (TRAP+ )-stained osteoclasts in an orthotopic model of local tumor growth after CHM1 knockdown, indicating that CHM1-mediated inhibition of osteomimicry might play a role in homing, colonization, and invasion into bone tissues. We further demonstrate that CHM1 enhanced the invasive potential of ES cells in vitro. This invasiveness was in part mediated via CHM1-regulated matrix metallopeptidase 9 expression and correlated with the observation that, in an xenograft mouse model, CHM1 was essential for the establishment of lung metastases. This finding is in line with the observed increase in CHM1 expression in patient specimens with ES lung metastases. Our results suggest that CHM1 seems to have pleiotropic functions in ES, which need to be further investigated, but appears to be essential for the invasive and metastatic capacities of ES.