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1.
Anticancer Res ; 40(11): 6327-6335, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33109570

RESUMO

BACKGROUND/AIM: Despite numerous studies, the etiology of chronic lymphocytic leukemia (CLL) remains unknown. A hypothesis of autoantigen stimulation in leukemic clone selection might explain 'stereotypy' of B-cell receptors. In healthy cells, cofilin-1 (CFL1) has multiple functions. Its role was described in several malignancies. The aim of this study was characterization of the role of CFL1 in CLL. Materialas and Methods: Cells from peripheral blood of 180 patients and 42 healthy volunteers (HVs) were isolated. Gene expression was assessed with reverse transcription polymerase chain reaction (RT-qPCR); western blot was performed for determination of protein level and activity. After silencing of CFL1 gene, cell ability for migration and chemotaxis was investigated with Transwell method. Post-silencing, apoptosis and cell cycle was determined by flow cytometry. RESULTS: In RT-qPCR, we observed significantly higher expression of CFL1. Higher activity of protein in CLL cells when compared to HVs was detected. Knock-down of CFL1 led to decreased chemotaxis and migration of CLL cells versus cells from HVs. Apoptosis was increased amongst cells with silenced CFL1 and correlated with higher proportion of cells in the G2/M phase. CONCLUSION: Significantly higher expression of CFL1 mRNA in CLL and higher protein activity might indicate high utilization of CFL1 in malignant cells, maintaining their viability, as its inhibition affected viability, cell-cycle progression and motility of leukemia cells.


Assuntos
Cofilina 1/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Transdução de Sinais , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Quimiotaxia/genética , Cofilina 1/genética , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismo
2.
Ann Hematol ; 99(11): 2629-2637, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32980890

RESUMO

Treatment of acute lymphoblastic leukemia (ALL) requires the combination of multiple drugs to integrate a complete remission. The different prognostic factors (age, leukocytes, risk, cytogenetic alterations) allow identifying those patients with a high risk of relapse, but there are few described factors that impact the induction response. The objective was to identify the utility of different risk factors (overexpression of the ABCB1 drug resistance gene, favorable response to steroids (FRS) and early response at day + 8 of treatment) on the percentage of complete remissions and overall survival. This is a prospective, observational study in adult patients with B-ALL without specific cytogenetic alterations, who started induction treatment based on a pretreatment with prednisone and subsequently vincristine (1.6 mg/m2 subcutaneous) plus daunorubicin (45 mg/m2 subcutaneously) on days + 1, + 8, + 15. The ABCB1 resistance gene was evaluated at diagnosis, the FRS at the end of the pretreatment and the early response during day + 8. A total of 53 adult patients diagnosed with ALL Philadelphia negative chromosome (Ph-), with immunophenotype B, with a normal karyotype, were studied. Cases with genetic abnormalities with a poor prognosis were excluded in order to reduce bias. The mean age was 48 years (range 17-68 years). 62.3% of patients were at high risk of relapse. When analyzing the risk factors, 30.2% showed high levels of the ABCB1 resistance gene, without showing an impact on the induction response (OR: 1.218, p = 0.743), but its overexpression was associated with a poor response to steroids as in the absence of early response. Individually, both the FRS (OR: 5.7, p = 0.004) and the absence of early response to day + 8 (OR: 6.42, p = 0.002) showed significance. By combining the different factors, having more than 2 was directly related to a failure (OR: 9.514, p = 0.000). The identification of factors such as FRS such as the persistence of blasts at the end of the first week of treatment is useful to identify patients at risk of failure in induction.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Quimioterapia de Indução , Proteínas de Neoplasias/biossíntese , Leucemia-Linfoma Linfoblástico de Células Precursoras , Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Adolescente , Adulto , Idoso , Daunorrubicina/administração & dosagem , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Prednisolona/administração & dosagem , Estudos Retrospectivos , Taxa de Sobrevida , Fatores de Tempo , Vincristina/administração & dosagem
3.
Nat Commun ; 11(1): 3455, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32661245

RESUMO

CRISPR-based genetic screening has revolutionized cancer drug target discovery, yet reliable, multiplex gene editing to reveal synergies between gene targets remains a major challenge. Here, we present a simple and robust CRISPR-Cas12a-based approach for combinatorial genetic screening in cancer cells. By engineering the CRISPR-AsCas12a system with key modifications to the Cas protein and its CRISPR RNA (crRNA), we can achieve high efficiency combinatorial genetic screening. We demonstrate the performance of our optimized AsCas12a (opAsCas12a) through double knockout screening against epigenetic regulators. This screen reveals synthetic sick interactions between Brd9&Jmjd6, Kat6a&Jmjd6, and Brpf1&Jmjd6 in leukemia cells.


Assuntos
Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleases/genética , Edição de Genes , Regulação Leucêmica da Expressão Gênica , Leucemia/genética , Animais , Proliferação de Células , Epigênese Genética , Biblioteca Gênica , Engenharia Genética , Genoma Humano , Células HEK293 , Humanos , Células K562 , Camundongos , Células NIH 3T3 , Domínios Proteicos , RNA Guia/genética
4.
Nat Commun ; 11(1): 2807, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32533074

RESUMO

The nuclear receptor binding SET domain protein 1 (NSD1) is recurrently mutated in human cancers including acute leukemia. We show that NSD1 knockdown alters erythroid clonogenic growth of human CD34+ hematopoietic cells. Ablation of Nsd1 in the hematopoietic system of mice induces a transplantable erythroleukemia. In vitro differentiation of Nsd1-/- erythroblasts is majorly impaired despite abundant expression of GATA1, the transcriptional master regulator of erythropoiesis, and associated with an impaired activation of GATA1-induced targets. Retroviral expression of wildtype NSD1, but not a catalytically-inactive NSD1N1918Q SET-domain mutant induces terminal maturation of Nsd1-/- erythroblasts. Despite similar GATA1 protein levels, exogenous NSD1 but not NSDN1918Q significantly increases the occupancy of GATA1 at target genes and their expression. Notably, exogenous NSD1 reduces the association of GATA1 with the co-repressor SKI, and knockdown of SKI induces differentiation of Nsd1-/- erythroblasts. Collectively, we identify the NSD1 methyltransferase as a regulator of GATA1-controlled erythroid differentiation and leukemogenesis.


Assuntos
Diferenciação Celular , Células Eritroides/metabolismo , Células Eritroides/patologia , Fator de Transcrição GATA1/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patologia , Adulto , Animais , Antígenos CD/metabolismo , Antígenos CD34/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eritroblastos/metabolismo , Fator de Transcrição GATA1/genética , Regulação Leucêmica da Expressão Gênica , Técnicas de Silenciamento de Genes , Hematopoese , Histona-Lisina N-Metiltransferase/genética , Humanos , Estimativa de Kaplan-Meier , Leucemia Eritroblástica Aguda/genética , Masculino , Camundongos , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores da Transferrina/metabolismo
5.
Toxicology ; 441: 152531, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32593706

RESUMO

Gene-regulatory networks reconstruction has become a very popular approach in applied biology to infer and dissect functional interactions of Transcription Factors (TFs) driving a defined phenotypic state, termed as Master Regulators (MRs). In the present work, cutting-edge bioinformatic methods were applied to re-analyze experimental data on leukemia cells (human myelogenous leukemia cell line THP-1 and acute myeloid leukemia MOLM-13 cells) treated for 6 h with two different Ribosome-Inactivating Proteins (RIPs), namely Shiga toxin type 1 (400 ng/mL) produced by Escherichia coli strains and the plant toxin stenodactylin (60 ng/mL), purified from the caudex of Adenia stenodactyla Harms. This analysis allowed us to identify the common early transcriptional response to 28S rRNA damage based on gene-regulatory network inference and Master Regulator Analysis (MRA). Both toxins induce a common response at 6 h which involves inflammatory mediators triggered by AP-1 family transcriptional factors and ATF3 in leukemia cells. We describe for the first time the involvement of MAFF, KLF2 and KLF6 in regulating RIP-induced apoptotic cell death, while receptor-mediated downstream signaling through ANXA1 and TLR4 is suggested for both toxins.


Assuntos
Redes Reguladoras de Genes/efeitos dos fármacos , Leucemia/metabolismo , Proteínas Inativadoras de Ribossomos/farmacologia , Linhagem Celular Tumoral , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Lectinas/farmacologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , N-Glicosil Hidrolases/farmacologia , Toxina Shiga I/farmacologia , Fatores de Transcrição/metabolismo
6.
Anticancer Res ; 40(5): 2457-2465, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32366389

RESUMO

The introduction of tyrosine kinase inhibitors (TKIs) directed against the catalytic activity of the ABL tyrosine kinase has considerably improved the outcome of chronic myeloid leukemia (CML) patients in the chronic phase of the disease. Indeed, these individuals currently show a life-expectancy comparable to those of healthy subjects. Currently, five TKIs (imatinib, dasatinib, nilotinib, bosutinib and ponatinib) are approved for the treatment of CML and can be used as first, second or further lines of treatment according to disease risk scores, patient comorbidities and the presence of known TKI resistance mechanisms. In fact, 15-20% of all CML patients fail to achieve optimal responses according to the current definitions of the European Leukemia Network and will require sequential TKI treatment to avoid disease progression. In this review, we present the state of art in several crucial areas of CML management by briefly: i) depicting the domain structure of the BCR-ABL1 oncoprotein; ii) describing pivotal data concerning TKI efficacy; iii) illustrating the diverse molecular mechanisms causing TKI resistance; and iv) summarizing new ABL1-directed therapeutic approaches that are presently under investigation.


Assuntos
Antineoplásicos/uso terapêutico , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/genética , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Proteínas Proto-Oncogênicas c-abl/genética , Resultado do Tratamento
7.
Leuk Res ; 94: 106383, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32460059

RESUMO

Metformin is an anti-diabetic drug known to have anticancer activity by inhibiting mechanistic target of rapamycin (mTOR); however, other molecular mechanisms may also be involved. In this study, we examined the effects of metformin on the activity of receptor tyrosine kinases of the TAM (TYRO3, AXL, and MERTK) family, which have important roles in leukemia cell growth. The results indicated that metformin suppressed the in vitro growth of four leukemia cell lines, OCI/AML2, OCI/AML3, THP-1, and K562, in a dose-dependent manner, which corresponded to the downregulation of the expression and phosphorylation of AXL and inhibition of its downstream targets such as phosphorylation of STAT3. Furthermore, metformin augmented the suppressive effects of a small-molecule AXL inhibitor TP-0903 on the growth of OCI/AML3 and K562 cells and prevented doxorubicin-induced AXL activation in K562 cells, which induces chemoresistance in leukemia cells, thus potentiating doxorubicin anti-proliferative effects. Given that metformin also downregulated expression of TYRO3 and phosphorylation of MERTK, these findings indicate that anti-leukemic effects exerted by metformin could be partly due to the inhibition of TAM kinases. Thus, metformin has a clinical potential for patients with leukemia cells positive for AXL and the other TAM proteins as well as activated mTOR.


Assuntos
Regulação para Baixo/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia , Metformina/farmacologia , Proteínas Proto-Oncogênicas/biossíntese , Receptores Proteína Tirosina Quinases/biossíntese , Humanos , Células K562 , Leucemia/tratamento farmacológico , Leucemia/enzimologia , Leucemia/patologia , Células THP-1
8.
Exp Hematol ; 85: 47-56.e2, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32360510

RESUMO

Recent experimental data suggest that the heterogeneity of chronic myeloid leukemia (CML) stem cells may be the result of the development of unique molecular events generating functional consequences in terms of the resistance and persistence of leukemic stem cells. To explore this phenomenon, we designed a single-cell transcriptome assay evaluating simultaneously the expression of 87 genes. Highly purified CD34+ cells from three CML patients at diagnosis were immobilized in microfluidic chips, and the expression of 87 genes was evaluated in each cell. This analysis identified a group of 13 highly connected genes including NANOG, POU5F1, LIN28A, and SOX2, representing on average 8.59% of the cell population analyzed. Bioinformatics analysis with the corrected matrix and t-distributed stochastic neighbor embedding (tSNE) algorithm identified four distinct clusters, and the pseudotime analysis confirmed the presence of seven stem cell states in the four clusters identified. ALOX5 expression was associated with the group of cells expressing the pluripotency markers. In in vitro analyses, two genes that were predicted to undergo similar regulation using pseudotime analysis (ALOX5 and FGFR) were found to be similarly inhibited by ponatinib, an FGFR inhibitor. Finally, in an independent cohort of CML patients, we found that pluripotency gene expression is a common feature of CD34+ CML cells at diagnosis. Overall, these experiments allowed identification of individual CD34+ cells expressing high levels of pluripotency genes at diagnosis, in which a continuum of transitional states were identified using pseudotime analysis. These results suggest that leukemic stem cell persistence in CML needs to be targeted simultaneously rather than using a single pathway.


Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteínas de Neoplasias/biossíntese , Células-Tronco Neoplásicas/metabolismo , Análise de Célula Única , Transcriptoma , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/patologia
9.
Gene ; 752: 144758, 2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32422235

RESUMO

Drugs targeting chromatin-modifying enzymes have entered clinical trials for myeloid malignancies, including INCB059872, a selective irreversible inhibitor of Lysine-Specific Demethylase 1 (LSD1). While initial studies of LSD1 inhibitors suggested these compounds may be used to induce differentiation of acute myeloid leukemia (AML), the mechanisms underlying this effect and dose-limiting toxicities are not well understood. Here, we used precision nuclear run-on sequencing (PRO-seq) and ChIP-seq in AML cell lines to probe for the earliest regulatory events associated with INCB059872 treatment. The changes in nascent transcription could be traced back to a loss of CoREST activity and activation of GFI1-regulated genes. INCB059872 is in phase I clinical trials, and we evaluated a pre-treatment bone marrow sample of a patient who showed a clinical response to INCB059872 while being treated with azacitidine. We used single-cell RNA-sequencing (scRNA-seq) to show that INCB059872 caused a shift in gene expression that was again associated with GFI1/GFI1B regulation. Finally, we treated mice with INCB059872 and performed scRNA-seq of lineage-negative bone marrow cells, which showed that INCB059872 triggered accumulation of megakaryocyte early progenitor cells with gene expression hallmarks of stem cells. Accumulation of these stem/progenitor cells may contribute to the thrombocytopenia observed in patients treated with LSD1 inhibitors.


Assuntos
Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Histona Desmetilases/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Animais , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA-Seq , Análise de Célula Única/métodos , Células-Tronco/metabolismo , Células THP-1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequenciamento Completo do Exoma/métodos
10.
Exp Hematol ; 85: 57-69, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32437908

RESUMO

Inhibition of the H3K79 histone methyltransferase DOT1L has exhibited encouraging preclinical and early clinical activity in KMT2A (MLL)-rearranged leukemia, supporting the development of combinatorial therapies. Here, we investigated two novel combinations: dual inhibition of the histone methyltransferases DOT1L and EZH2, and the combination with a protein synthesis inhibitor. EZH2 is the catalytic subunit in the polycomb repressive complex 2 (PRC2), and inhibition of EZH2 has been reported to have preclinical activity in KMT2A-r leukemia. When combined with DOT1L inhibition, however, we observed both synergistic and antagonistic effects. Interestingly, antagonistic effects were not due to PRC2-mediated de-repression of HOXA9. HOXA cluster genes are key canonical targets of both KMT2A and the PRC2 complex. The independence of the HOXA cluster from PRC2 repression in KMT2A-r leukemia thus affords important insights into leukemia biology. Further studies revealed that EZH2 inhibition counteracted the effect of DOT1L inhibition on ribosomal gene expression. We thus identified a previously unrecognized role of DOT1L in regulating protein production. Decreased translation was one of the earliest effects measurable after DOT1L inhibition and specific to KMT2A-rearranged cell lines. H3K79me2 chromatin immunoprecipitation sequencing patterns over ribosomal genes were similar to those of the canonical KMT2A-fusion target genes in primary AML patient samples. The effects of DOT1L inhibition on ribosomal gene expression prompted us to evaluate the combination of EPZ5676 with a protein translation inhibitor. EPZ5676 was synergistic with the protein translation inhibitor homoharringtonine (omacetaxine), supporting further preclinical/clinical development of this combination. In summary, we discovered a novel epigenetic regulation of a metabolic process-protein synthesis-that plays a role in leukemogenesis and affords a combinatorial therapeutic opportunity.


Assuntos
Epigênese Genética , Regulação Leucêmica da Expressão Gênica , Rearranjo Gênico , Leucemia Mieloide Aguda/metabolismo , Biossíntese de Proteínas , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Proteína de Leucina Linfoide-Mieloide/genética , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo
11.
Exp Hematol ; 86: 21-27.e2, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32437909

RESUMO

Our previous study revealed that expression of G protein-coupled receptor 68 (GPR68) was upregulated in MDSL cells, a cell line representing myelodysplastic syndromes (MDS), in response to lenalidomide (LEN), and mediated a calcium/calpain proapoptotic pathway. Isx, a GPR68 agonist, enhanced the sensitivity to LEN in MDSL cells. The fact that Isx is not a U.S. Food and Drug Administration-approved drug prompts us to look for alternative candidates that could enhance the sensitivity of LEN in MDS as well as other hematologic malignancies, such as acute myeloid leukemia (AML). In the study described here, we found that regulator of calcineurin 1 (RCAN1), an endogenous inhibitor of calcineurin (CaN), was upregulated in MDSL cells in response to LEN, possibly through degradation of IKZF1. Consistently, cyclosporin (Cys), a pharmacological inhibitor of CaN, inhibited the activity of CaN and induced apoptosis in MDSL cells, indicating that CaN provided a prosurvival signal in MDSL cells. In addition, Cys enhanced the cytotoxic effect of LEN in MDS/AML cell lines as well as primary bone marrow cells from MDS patients and AML patient-derived xenograft models. Intriguingly, pretreatment with LEN reversed the suppressive effect of Cys on T-cell activation. Our study suggests a novel mechanism of action of LEN in mediating cytotoxicity in MDS/AML via upregulation of RCAN1, thus inhibiting the CaN prosurvival pathway. Our study also suggests that Cys enhances the sensitivity to LEN in MDS/AML cells without compromising T-cell activation.


Assuntos
Ciclosporina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Lenalidomida/farmacologia , Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclosporina/agonistas , Proteínas de Ligação a DNA/biossíntese , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Fator de Transcrição Ikaros/biossíntese , Lenalidomida/agonistas , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Proteínas Musculares/biossíntese , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Proteínas de Neoplasias/biossíntese , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Proc Natl Acad Sci U S A ; 117(22): 12332-12340, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32424097

RESUMO

Double knockout of the two miR-15/16 loci in mouse resulted in the development of acute myeloid leukemia (AML). This result suggested that, at least, a fraction of human AMLs could be due to a similar mechanism. We analyzed the role of the two miR-15/16 clusters in 93 myelodysplastic syndrome (MDS) patients divided in three subgroups: patients with MDS, patients with MDS before transforming into AML (MDS-T), and patients with AML evolving from MDS (MDS-AML). Then, we tested 139 AML cases and 14 different AML cell lines by assessing microRNA (miRNA) expression, target protein expression, genetic loss, and silencing. MDS-T and MDS-AML patients show a reduction of the expression of miR-15a/-15b/-16 compared to MDS patients. Each miRNA can significantly predict MDS and MDS-T groups. Then, 79% of primary AMLs show a reduced expression of miR-15a and/or miR-15b. The expression of miR-15a/-15b/-16 significantly stratified AML patients in two prognostic classes. Furthermore, 40% of AML cell lines showed a combined loss of the expression of miR-15a/-15b and overexpression of their direct/indirect targets. As potential mechanisms involved in the silencing of the two miR-15/16 loci, we identified a genetic loss of miR-15a and miR-15b and silencing of these two loci by methylation. We identified a potential driver oncogenic role in the loss of expression of both miR-15/16 clusters in the progression of MDS into AML and in AML pathogenesis. The stratification of AML patients, based on miR-15/16 expression, can lead to targeted and combination therapies for the treatment of this incurable disease.


Assuntos
Leucemia Mieloide Aguda/genética , MicroRNAs/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Progressão da Doença , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade
13.
Exp Hematol ; 85: 8-12, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32283132

RESUMO

Approximately 10% of the human transcriptome is composed of circular RNAs (circRNAs). These are non-coding RNA molecules in which a covalent bond between the 3' and 5' forms a stable circular loop. Herein, we profiled the expression of 13,368 cricRNAS in 21 patients with chronic lymphocytic leukemia (CLL). Regardless of clinical, genetic, or prognostic characteristics, CLL cells share a unique expression profile distinguishable from that of normal B cells. Specifically, 859 circRNAs from 592 genes were differentially expressed (fold change ≥2 and false discovery rate ≤0.05). Whether dysregulation of circRNAs contributes to the pathogenesis of CLL remains to be determined.


Assuntos
Biologia Computacional , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/metabolismo , RNA Circular/biossíntese , RNA Viral/biossíntese , Feminino , Seguimentos , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , RNA Circular/genética , RNA Viral/genética
14.
Cancer Sci ; 111(7): 2361-2373, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32314454

RESUMO

To elucidate dynamic changes in native BCR-ABL and alternatively spliced tyrosine kinase inhibitor (TKI)-resistant but function-dead BCR-ABLIns35bp variant, following commencement or discontinuation of TKI therapy, each transcript was serially quantified in patients with chronic myeloid leukemia (CML) by deep sequencing. Because both transcripts were amplified together using conventional PCR system for measuring International Scale (IS), deep sequencing method was used for quantifying such BCR-ABL variants. At the initial diagnosis, 7 of 9 patients presented a small fraction of cells possessing BCR-ABLIns35bp , accounting for 0.8% of the total IS BCR-ABL, corresponding to actual BCR-ABLIns35bp value of 1.1539% IS. TKI rapidly decreased native BCR-ABL but not BCR-ABLIns35bp , leading to the initial increase in the proportion of BCR-ABLIns35bp . Thereafter, both native BCR-ABL and BCR-ABLIns35bp gradually decreased in the course of TKI treatment, whereas small populations positive for TKI-resistant BCR-ABLIns35bp continued fluctuating at low levels, possibly underestimating the molecular response (MR). Following TKI discontinuation, sequencing analysis of 54 patients revealed a rapid relapse, apparently derived from native BCR-ABL+ clones. However, IS fluctuating at low levels around MR4.0 marked a predominant persistence of cells expressing function-dead BCR-ABLIns35bp , suggesting that TKI resumption was unnecessary. We clarified the possible mechanism underlying mis-splicing BCR-ABLIns35bp , occurring at the particular pseudo-splice site within intron8, which can be augmented by TKI treatment through inhibition of RNA polymerase II phosphorylation. No mutations were found in spliceosomal genes. Therefore, monitoring IS functional BCR-ABL extracting BCR-ABLIns35bp would lead us to a correct evaluation of MR status, thus determining the adequate therapeutic intervention.


Assuntos
Processamento Alternativo , Proteínas de Fusão bcr-abl/genética , Mutação , Inibidores de Proteínas Quinases/farmacologia , RNA Polimerase II/metabolismo , Adulto , Idoso , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Loci Gênicos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Íntrons , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Masculino , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/uso terapêutico , RNA Polimerase II/antagonistas & inibidores , Análise de Célula Única
15.
Nat Commun ; 11(1): 2026, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332729

RESUMO

The cell-context dependency for RNA binding proteins (RBPs) mediated control of stem cell fate remains to be defined. Here we adapt the HyperTRIBE method using an RBP fused to a Drosophila RNA editing enzyme (ADAR) to globally map the mRNA targets of the RBP MSI2 in mammalian adult normal and malignant stem cells. We reveal a unique MUSASHI-2 (MSI2) mRNA binding network in hematopoietic stem cells that changes during transition to multipotent progenitors. Additionally, we discover a significant increase in RNA binding activity of MSI2 in leukemic stem cells compared with normal hematopoietic stem and progenitor cells, resulting in selective regulation of MSI2's oncogenic targets. This provides a basis for MSI2 increased dependency in leukemia cells compared to normal cells. Moreover, our study provides a way to measure RBP function in rare cells and suggests that RBPs can achieve differential binding activity during cell state transition independent of gene expression.


Assuntos
Diferenciação Celular/genética , Células-Tronco Hematopoéticas/patologia , Leucemia/genética , Células-Tronco Neoplásicas/patologia , Proteínas de Ligação a RNA/metabolismo , Adenosina Desaminase/genética , Animais , Sítios de Ligação/genética , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes , Células HEK293 , Humanos , Leucemia/sangue , Leucemia/patologia , Camundongos , Camundongos Knockout , Cultura Primária de Células , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , RNA-Seq , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
16.
Proc Jpn Acad Ser B Phys Biol Sci ; 96(3): 107-121, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32161209

RESUMO

During the past decade, substantial progress has been made in the field of the genetics of myelodysplastic syndromes (MDS). These comprise a group of chronic myeloid neoplasms with abnormal cell morphology and progression to acute myeloid leukemia (AML), where revolutionary sequencing technologies have played a major role. Through extensive sequencing of a large number of MDS genomes, a comprehensive registry of driver mutations involved in the pathogenesis of MDS has been revealed, along with their impacts on clinical phenotype and prognosis. The most frequently affected molecules are involved in DNA methylations, chromatin modification, RNA splicing, transcription, signal transduction, cohesin regulation, and DNA repair. These mutations show strong positive and negative correlations with each other, suggesting the presence of functional interactions between mutations, which dictate disease progression. Because these mutations are associated with disease phenotype, drug response, and clinical outcomes, it is essential to be familiar with MDS genetics not only for better understanding of MDS pathogenesis but also for management of patients.


Assuntos
DNA de Neoplasias/genética , Síndromes Mielodisplásicas/classificação , Síndromes Mielodisplásicas/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Metilação de DNA , Reparo do DNA , DNA de Neoplasias/metabolismo , Progressão da Doença , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide de Fase Crônica/genética , Mutação , Fenótipo , Prognóstico , Processamento de RNA , Análise de Sequência de DNA , Transdução de Sinais
17.
Med Sci Monit ; 26: e921288, 2020 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-32146479

RESUMO

BACKGROUND Leukemia is common in aging adults and has very high mortality worldwide. The present study was designed to investigate the therapeutic efficacy of miR-18a inhibitor against WEHI-3 and THP-1 leukemia cells. MATERIAL AND METHODS The changes in miR-18a inhibitor-transfected WEHI-3 and THP-1 cell proliferative potential was measured by use of the Cell Counting Kit-8 assay. Apoptotic changes were analyzed by electron microscopy, and evaluation of PI3K, AKT, mTOR, and PTEN expression was assessed by RT-qPCR assay. RESULTS Transfection of miR-18a inhibitor significantly (P<0.05) suppressed the proliferative potential of WEHI-3 and THP-1 cells. The WEHI-3 cells showed the presence of characteristic apoptotic bodies on transfection with miR-18a inhibitor at 48 h. Flow cytometry showed that miR-18a inhibitor transfection significantly (P<0.05) increased the WEHI-3 cell percentage in G1 phase. The transfection of miR-18a inhibitor significantly (P<0.05) promoted apoptosis in WEHI-3 cells. In WEHI-3 cells, miR-18a inhibitor transfection markedly suppressed the expression of PI3K, AKT, and mTOR mRNA. The expression of PTEN mRNA was significantly (P<0.05) upregulated by miR-18a inhibitor transfection in WEHI-3 cells. CONCLUSIONS The present study investigated the therapeutic efficacy of miR-18a inhibitor against WEHI-3 and THP1 leukemia cells. The study demonstrated that miR-18a inhibitor suppressed the proliferative potential of WEHI-3 and THP1 cells and activated apoptotic process through upregulation of PTEN mRNA expression. Therefore, miR-18a inhibitor can be of therapeutic importance for the treatment of leukemia.


Assuntos
Proliferação de Células/efeitos dos fármacos , Leucemia/patologia , MicroRNAs/antagonistas & inibidores , PTEN Fosfo-Hidrolase/metabolismo , Apoptose , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia/tratamento farmacológico , Células THP-1 , Transfecção , Regulação para Cima
18.
Leuk Res ; 91: 106316, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32114371
19.
Oncol Res ; 28(4): 357-369, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32183929

RESUMO

It has been reported that kindlin-3 expression is closely associated with progression of many cancers and microRNA (miRNA) processing. However, the effects and precise mechanisms of kindlin-3 in acute myeloid leukemia (AML) have not been well clarified. Our study aimed to explore the interaction between kindlin-3 and miR-4792 in AML. In our study, we found that the expression of kindlin-3 was dramatically increased in AML samples and cell lines, and the miR-4792 level was significantly downregulated. Interestingly, the low miR-4792 level was closely associated with upregulated kindlin-3 expression in AML samples. Moreover, introduction of miR-4792 dramatically suppressed proliferation and invasion and induced apoptosis of AML cells. We demonstrated that miR-4792 could directly target kindlin-3 by using both bioinformatics analysis and luciferase reporter assay. In addition, kindlin-3 silencing had similar effects with miR-4792 overexpression on AML cells. Overexpression of kindlin-3 in AML cells partially reversed the inhibitory effects of miR-4792 mimic. miR-4792 inhibited cell proliferation and invasion and induced apoptosis of AML cells by directly downregulating kindlin-3 expression, and miR-4792 targeting kindlin-3 was responsible for the regulation of the proliferation, invasion, and apoptosis of AML cells.


Assuntos
Apoptose/genética , Proliferação de Células/genética , Leucemia Mieloide Aguda/genética , Proteínas de Membrana/genética , MicroRNAs/genética , Proteínas de Neoplasias/genética , Regiões 3' não Traduzidas , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/patologia , Invasividade Neoplásica/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima
20.
Sci China Life Sci ; 63(5): 675-687, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32170627

RESUMO

Transposable elements (TEs) have been shown to have important gene regulatory functions and their alteration could lead to disease phenotypes. Acute myeloid leukemia (AML) develops as a consequence of a series of genetic changes in hematopoietic precursor cells, including mutations in epigenetic factors. Here, we set out to study the gene regulatory role of TEs in AML. We first explored the epigenetic landscape of TEs in AML patients using ATAC-seq data. We show that a large number of TEs in general, and more specifically mammalian-wide interspersed repeats (MIRs), are more enriched in AML cells than in normal blood cells. We obtained a similar finding when analyzing histone modification data in AML patients. Gene Ontology enrichment analysis showed that genes near MIRs in open chromatin regions are involved in leukemogenesis. To functionally validate their regulatory role, we selected 19 MIR regions in AML cells, and tested them for enhancer activity in an AML cell line (Kasumi-1) and a chronic myeloid leukemia (CML) cell line (K562); the results revealed several MIRs to be functional enhancers. Taken together, our results suggest that TEs are potentially involved in myeloid leukemogenesis and highlight these sequences as potential candidates harboring AML-associated variation.


Assuntos
Elementos de DNA Transponíveis/genética , Elementos Facilitadores Genéticos/genética , Regulação Leucêmica da Expressão Gênica/genética , Leucemia Mieloide Aguda/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Cromatina/genética , Ontologia Genética , Genoma Humano , Humanos , Camundongos , Modelos Biológicos , Motivos de Nucleotídeos , Fatores de Transcrição
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