BRD4 inhibition enhances azacitidine efficacy in acute myeloid leukemia and myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell-based disorders characterized by ineffective hematopoiesis, increased genomic instability and a tendency to progress toward acute myeloid leukemia (AML). MDS and AML cells present genetic and epigenetic abnormalities and, due to the heterogeneity of thesemolecular alterations, the current treatment options remain unsatisfactory. Hypomethylating agents (HMA), especially azacitidine, are the mainstay of treatment for high-risk MDS patients and HMA are used in treating elderly AML. The aim of this study was to investigate the potential role of the epigenetic reader bromodomain-containing protein-4 (BRD4) in MDS and AML patients. We identified the upregulation of the short variant BRD4 in MDS and AML patients, which was associated with a worse outcome of MDS. Furthermore, the inhibition of BRD4 in vitro with JQ1 or shRNA induced leukemia cell apoptosis, especially when combined to azacitidine, and triggered the activation of the DNA damage response pathway. JQ1 and AZD6738 (a specific ATR inhibitor) also synergized to induce apoptosis in leukemia cells. Our results indicate that the BRD4-dependent transcriptional program is a defective pathway in MDS and AML pathogenesis and its inhibition induces apoptosis of leukemia cells, which is enhanced in combination with HMA or an ATR inhibitor9CNPQ - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPESP – Fundação de Amparo à Pesquisa Do Estado De São Paulonão tem2011/51959-

BRD4 Inhibition Enhances Azacitidine Efficacy in Acute Myeloid Leukemia and Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell-based disorders characterized by ineffective hematopoiesis, increased genomic instability and a tendency to progress toward acute myeloid leukemia (AML). MDS and AML cells present genetic and epigenetic abnormalities and, due to the heterogeneity of these molecular alterations, the current treatment options remain unsatisfactory. Hypomethylating agents (HMA), especially azacitidine, are the mainstay of treatment for high-risk MDS patients and HMA are used in treating elderly AML. The aim of this study was to investigate the potential role of the epigenetic reader bromodomain-containing protein-4 (BRD4) in MDS and AML patients. We identified the upregulation of the short variant BRD4 in MDS and AML patients, which was associated with a worse outcome of MDS. Furthermore, the inhibition of BRD4 in vitro with JQ1 or shRNA induced leukemia cell apoptosis, especially when combined to azacitidine, and triggered the activation of the DNA damage response pathway. JQ1 and AZD6738 (a specific ATR inhibitor) also synergized to induce apoptosis in leukemia cells. Our results indicate that the BRD4-dependent transcriptional program is a defective pathway in MDS and AML pathogenesis and its inhibition induces apoptosis of leukemia cells, which is enhanced in combination with HMA or an ATR inhibitor

Comparison of different methods to overexpress large genes

Gain-of-function of very large transgene constructs can lead to genetic perturbations, providing researchers with the alternative of a powerful tool to identify pathway components which remain undetected when using traditional loss-of-function analysis. To promote longer-term expression, various systems for transgene integration have been developed, however large cDNA sequences are often difficult to clone into size-limited expression vectors. We attempted to overexpress ARHGAP21, a 5.874 kb gene, using different methodologies as plasmid, lentiviral and Sleeping Beauty (SB) transposon based gene transfer. Using lentiviral based transduction; an enormous amount of lentiviral supernatant was produced to obtain a satisfactory titration after double ultracentrifugation. However, U937 transduced cells showed only 50% of gene expression increase, which vanished after 5 days. SB transposon system application to overexpress ARHGAP21 was a complete success. Nucleofecting SB-based vector plus SB100x transposase vector resulted in an expressive increase of gene and protein expression. Furthermore, the overexpression was maintained even after freezing and thawing processes. In conclusion, our work shows that the SB transposon system is the best choice for those seeking a stable and high gene expression. Once the overexpression is achieved, freezing cells and using them for a long time becomes possible

Characterization of cytotoxic heat-stable enterotoxin (ST-L) produced by Escherichia coli isolated from drinking water

Orientador: Domingos da Silva LeiteDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Estudos das propriedades de virulência de Escherichia coli associadas às infecções intestinais são importantes para compreender seus mecanismos diarreiogênicos. Em um trabalho anterior, realizado em nosso laboratório descrevemos um fator de virulência no sobrenadante de E. coli isoladas de água de consumo na cidade de Ouro Preto, MG, com atividades enterotóxica e citotóxica. Este fator foi denominado enterotoxina termo-estável ST-like (ST-L). A ST-L foi purificada a partir dos sobrenadantes de cultura de amostras de E.coli cultivadas em meio CAYE modificado, fracionado com sulfato de amônio e por ultrafiltração e concentrado por liofilização. Este material foi submetido às cromatografias de exclusão molecular e fase reversa, em sistema HPLC. A ST-L purificada mostrou, pela análise eletroforética em 2D, ponto isoelétrico (pI) em 6,19 e massa molecular de aproximadamente 2kDa. A atividade biológica pelos tratamentos com enzimas tripsina, proteinase K, ribonuclease ou pepsina. A citotoxicidade da ST-L manteve-se inalterada sob variações de pH (3 a 9) e tratamento com solventes orgânicos. A toxina resistiu ao aquecimento à 100ºC mostrando ser termoestável. A caracterização biológica da ST-L mostrou que várias linhagens celulares foram sensíveis aos seus efeitos citotóxicos, sofrendo alterações celulares e nucleares, em até 24h, com a formação de vacúolos citoplasmáticos e morte celular, conforme verificado pelos testes de viabilidade celular dos lisossomos, mitocôndrias, membranas celulares e quantificação da população celular das linhagens HEp-2, Vero, CaCo-2 e HT-29. A ST-L não induziu acúmulo de fluídos no teste de alça ligada em intestino de coelhos induzindo, contudo, acúmulo de fluidos intestinais no teste de camundongos recém-nascidos (Dean). Foi possível determinar que a toxina STL encontra-se codificada num plasmídeo e apresenta uma região conservada de alta similaridade com estA2, o que justifica a presença de efeito enterotóxico no teste de Dean. No entanto, não foi possível determinar a região ativa para a atividade citotóxica da ST-L. Em função das características apresentadas sugere-se a toxina ST-L seja uma variante do grupo das enterotoxinas termoestáveis (ST) com atividade citotóxica produzida por E. coli diarreiogênicas.Abstratc: Studies on the virulence properties of Escherichia coli associated with intestinal infections are important for the understanding of diarrheic diseases mechanisms. In a previous work we have described a factor presenting enterotoxic and cytotoxic activities in the culture supernatants of E. coli isolated from drinking water in the city of Ouro Preto, MG. This factor was called as thermo stable enterotoxin - like (ST-L) and it was purified from cultured supernatants produced with modified CAYE medium, fractionated with ammonium sulfate, being subsequently ultrafiltrated, subjected to molecular exclusion chromatography and then to reversed-phase chromatography in HPLC-system. The ST-L showed to be pure by the electrophoretic analysis in the bidimensionl electrophoresis (2D), being the isoelectric point (pI) of 6.19 and the molecular mass of approximately 2kDa. The toxin can not be inactivated by enzimes like trypsin, proteinase K, ribonuclease or pepsin. The cytotoxic activity of ST-L does not change whith pH variations (3 - 9), does not change after treatment with organic solvents and resists heating treatments of 100°C showing it to be heat-stable. Biological characterization of the toxin showed, through the tests of cell viability with the ST-L, that several cell lines were sensitive to the cytotoxin action, with major cellular and nuclear changes observed within 24 hour assays, presenting cytoplasmic vacuoles and cell death, what was verified by tests of cell viability of lysosomes, mitochondria, cell membranes and quantification of the cell population on cell lines like HEp-2, Vero, CaCo-2 and HT-29. The rabbits ileal intestinal loop tests, performed with the ST-L, did not show positive results, but the toxin acts by inducing intestinal fluid accumulation into the newborn mice test (Dean). Through molecular characterization tests, we found that the gene enconding for the toxin ST-L is in a plasmid and has a conserved region with high similarity to estA2, what justifies the presence of the positive enterotoxic result in the Dean test. However, it was not possible to determine the fragment responsible for cytotoxic activity of ST-L. According to the characteristics presented we suggest that the toxin-ST-L is a variant group of the heat-stable enterotoxin (ST) with cytotoxic activity produced by the diarrheagenic E. coli.MestradoMicrobiologiaMestre em Genética e Biologia Molecula

Artemisinins induce endoplasmic reticulum stress in acute leukaemia cells in vitro and in vivo

Abstract Loss of endoplasmic reticulum (ER) homeostasis leads to ER stress, thus prolonged activation can lead to apoptosis. Herein, artesunate (ART) induced ER stress in leukaemia cells, resulting in eIF2α phosphorylation, activation of transcription factor 4, subsequent CHOP upregulation and XBP1 splicing. Furthermore, in vitro cyclin/CDKs reduction induced G1‐phase arrest. An in vivo xenograft model showed a consistent pattern of ART in reducing tumour burden, supporting roles in the UPR pathway, which we speculate could lead to apoptosis by NOXA activation. Moreover, ART were capable of increasing the survival of mice. Taken together, our data indicate that ART may represent an interesting weapon to fight leukaemia

SEMA3A partially reverses VEGF effects through binding to neuropilin-1

Cross-talk between hematopoietic stem cells (HSCs) and bone marrow stromal cells (BMSCs) is essential for HSCs regulation and leukemogenesis. Studying bone marrow of myelodysplasia patients, a pre-leukemic condition, we found mRNA overexpression of vascular endothelial growth factor A (VEGFA) in CD34+ HSCs and semaphorin 3A (SEMA3A) in BMSCs. To better understand the role of VEGFA and SEMA3A in leukemogenesis, we recruited 30 myelodysplastic syndrome (MDS) patients, 29 acute myeloid leukemia (6 secondary to MDS) patients and 12 controls. We found higher VEGFA expression in de novo AML patients (without prior MDS) group (p = 0.0073) and higher SEMA3A expression in all BMSCs patient's samples compared to control group. We then overexpressed VEGFA in an acute myelogenous leukemia cell line, KG1 cells, and in normal CD34+ cells. This overexpression increased KG1 (p = 0.045) and CD34+ cell (p = 0.042) viability and KG1 (p = 0.042) and CD34+ cell (p = 0.047) proliferation. Moreover, KG1 and CD34+ cells overexpressing VEGFA also had increased proliferation when co-cultured with human marrow stromal HS5 cells (p = 0.045 and p = 0.02, respectively). However, co-culture of these transformed cells with HS5 cells overexpressing SEMA3A reduced KG1 (p = 0.004) and CD34+ (p = 0.009) proliferation. Co-culture of KG1 transformed cells with HS27 cells overexpressing SEMA3A reduced KG1 proliferation as well (p = 0.01). To investigate whether the dominant SEMA3A effect over VEGFA could be due to competition for neuropilin1 receptor (NRP1), we performed immunoprecipitation with anti-NRP1 antibody of cell extracts of co-cultured KG1 and HS5 cells, induced or not by VEGFA and SEMA3A recombinant proteins. Results showed a preferential association of NRP1 with SEMA3A, suggesting that SEMA3A can partially reverse the effects caused by the VEGFA preventing its binding with the NRP1 receptor. Since both hematopoietic cells, leukemic and normal, showed similar behavior, we suppose that the attempt to reversion of VEGF effects by SEMA3A is a homeostatic phenomenon in the hematopoietic niche. Finally, we conclude that VEGFA overexpression confers AML cell advantages and SEMA3A may partially reverse this effect; thus, SEMA3A protein combined with VEGFA inhibitors could be beneficial for AML treatment

Production of dendritic cell vaccines using different methods with equivalent results: Implications for emerging centers

Introduction: Dendritic cell (DC) vaccines have demonstrated good efficacy in preventing relapse and in increasing survival of patients affected by a variety of both solid and hematological tumors. Most protocols used to generate these cells involve the automated separation of peripheral blood monocytes from patients. This approach requires specialized equipment, which elevates the cost of this type of therapy, potentially limiting the widespread access to patients. Method: In this study, we compare the yield and quality of dendritic cells generated from monocytes and isolated by an automated method or by manual methods using gradient centrifugation. Results: The results demonstrate the equivalence of the 3 methods in relation to the yield and final quality of the product, however with considerable differences between the costs of these procedures. In addition, this study also demonstrates the feasibility of the antigenic pulse with autologous tumor cell lysates, constituting a source of antigens, not only easily obtained and manipulated, but also specific to the patient's tumor. Conclusion: These findings may have important implications for emerging centers interested in using this medical approach and potentially increase the access of a greater number of patients to this therapeutic option