ALBERT

Description: Abstract 2215 Poster Board II-192 The exact action mechanism of the protein BCR-ABL is unknown, studies in vitro and in animal models showed that the activity of protein tyrosine kinase (TK), by itself, is sufficient to cause the development of CML. Thus, the progression of the disease to accelerated phase or blast crisis may be associated with genomic instability. The use of methods to detect the difference in gene expression between CML and normal granulocytes could bring new insights in the understanding of these complex mechanisms, highlighting new therapeutic targets for the CML treatment. Evaluation of the differential gene expression between CML and normal granulocytes using the Subtractive Suppression Hybridization (SSH) and investigation of the involvement of a set of genes in modulating the development of CML. The SSH libraries were constructed using a pool of granulocytes RNA's extracted from CML patients and from healthy blood donors. Real-Time (RT-PCR) was used to validate the results and evaluation of gene expression in granulocytes and in mononuclear cells in the pool of patients and controls used for the construction of libraries. The expression of the RUNX1 gene, whose expression was the most differential between the libraries, was evaluated in mononuclear cells of patients with CML using RT-PCR. The comparison between granulocytes of CML patients and control granulocytes showed 39 genes exclusively expressed in CML and 169 genes in controls. For validation, we evaluated the expression of genes RUNX1, KPNA6, TOB1, SEPT5, CDC42SE, ITCH, MIER and GP1BA by RT-PCR in the pool of patients and controls used for the construction of libraries. Among these genes, the RUNX1 gene showed substantial difference, and was also studied in mononuclear cells from 20 patients with CML in different stages of the disease and using different types of treatment with TK inhibitors. The expression of this gene was highly altered in patients in advanced phase of disease when compared with patients who are responding to treatment. The genes identified in this study may be related to the development and progression of CML. Among them, the RUNX1 gene was shown to be one of the most promising, since the occurrence of chromosomal translocations in this gene has been associated with different types of acute leukemia – however its association with CML is not yet clear. The results showed a relationship between the expression of this gene and progression of the disease, beyond the identification of several genes that may lead to a better understanding of CML and help in identifying new therapeutic targets. This work was supported by FAPESP. Disclosures: Off Label Use: Development of TKI in the treatment of CML.

Description: Introduction: Satisfactory response is present for the majority of chronic myeloid leukemia (CML) patients (pts) in chronic phase (CP) treated with tyrosine kinase inhibitors (ITK) . However, some pts exhibit suboptimal response or treatment failure. The probability of achieving optimal response may be related with several factors. The oxidative stress modulation is tightly related with the physiopathology of various hematologic diseases and can cause cell death, apoptosis and necrosis. Peroxiredoxins (Prdx) are a family of multifunctional antioxidant thioredoxin-dependent peroxidases that protect cells against oxidative stress and modulate signaling cell proliferation pathways and may influence the metabolism of ITKs.The aim of this study was to analyze PRDX1, PRDX2 and PRDX6 levels of CML pts and correlate with cytogenetics and molecular responses. Methods: PRDX1, PRDX2 and PRDX6 expression was evaluated in 20 blood donors, 18 newly diagnosed CML pts and 22 previously treated pts. Pts were treated with imatinib 400-600mg in first line. Samples were collected from peripheral blood at diagnosis or during treatment and RNA samples were submitted to the synthesis of complementary DNA (cDNA) using the kit RevertAid™ HMinus First Strand cDNA Synthesis Kit (Fermentas, Life Sciences). For cDNA synthesis, 3 ug of RNA was used and peroxiredoxins expression was evaluated by real-time PCR with Syber Green (Applied Biosystems) and endogenous (β-Actina and GAPDH) controls. The results were analyzed using 2-ΔΔCT. Statistical analysis were made by using Mann Withney’s T test. Cytogenetic analysis was performed at diagnosis, 3, 6, 12 and 18 months after starting therapy and then every 12-24 months thereafter if CCR was achieved. BCR-ABL transcripts were measured in peripheral blood at 3-month intervals using quantitative RQ-PCR. Results were expressed as BCR-ABL/ABL ratio, with conversion to the international scale (IS). Major molecular response (MMR) was defined as a transcript level ≤ 0.1% (IS). Results: 40 CML pts, 55% male, median age of 53 years (23-84) were evaluated, 60% in chronic phase (CP), 30% in accelerated phase (AP) and 10% in blast crisis (BC). The mean of PRDX transcript levels in the total group was (PRDX1: 0.006 and 10.10 / PRDX2: 0.002 and 16.26 / PRDX6: 0.003 and 49.97) respectively (PRDX1: 1.2 / PRDX2: 0.9 / PRDX6: 15.36). The results showed that there are a significantly difference (p

Description: Abstract 4797 Background: Extensive studies have led to a considerable understanding of the cellular and molecular control of haemoglobin production during red blood cell differentiation, however, identification of the genes expressed as part of the erythroid differentiation programme remains an important goal because of the insights that these data will bring to erythrocyte biology and disease. Previous results using SAGE identified 93 differentially-expressed genes during erythroid development. One of these genes, EYA3, a homologue gene of Eyes Absent 3 in Drosophila, is a transcription cofactor with intrinsic phosphatase activity and its expression was observed to be high at the end of CD34+ cell differentiation and in human bone marrow. Aim: To evaluate globin gene, fetal hemoglobin (HbF) expressions and apoptosis levels in the erythroleukemic K562 cell line after EYA3 gene silencing and induction with hemin. Methods: Four different cultures from human K562 cells (1×105cells/mL in DMEM, 10% FBS, penicillin/streptomycin, 5% CO2, 37°C) were transfected with control or EYA3 knockdown lentiviruses (MOI=2.5). After proliferation and selection of successfully transfected cells with puromicin (2.0 ug/mL), cells were treated with 30μM hemin and collected after 0, 24, 48, 72 and 96h for gene expression and flow cytometry analyses. EYA3, LXN, α, and g-gene expression was measured by qRT-PCR and normalized using the Genorm program. HbF expression and apoptosis were evaluated by flow cytometry. Results: Analysis of globin gene expression showed that α-globin gene was downregulated in EYA3 silenced K562 culture cells compared with the control culture in 72h after hemin addition (1.791±0.1735; 0.7404±0.1709, respectively, **P

Description: Abstract 2066 Hereditary persistence of fetal hemoglobin (HPFH) is a condition that prevents hemoglobin switching and the consequent silencing of the gamma globin genes, resulting in continued hemoglobin (Hb) F synthesis in adults. Two types of HPFH are responsible for this phenotype: deletional HPFH – deletions in the end of the beta globin locus – and non-deletional HPFH (ndHPFH) – single point mutations in the proximal promoter of both gamma globin genes. Sickle cell anemia patients or beta-thalassemia patients that present HPFH show high levels of HbF that are associated with less severe clinical course in these diseases. The development of new therapies based on the reactivation of gamma globin expression may be important for the treatment of these patients. The Brazilian ndHPFH type is characterized as a C→G substitution in the A gamma globin promoter at position –195 and the molecular mechanism responsible for the reactivation of this gene in the Brazilian ndHPFH type remains unclear. In contrast to the British ndHPFH type (-198), where the mechanism responsible for the increase of HbF levels is mediated by the raising in the affinity for the Sp1 transcription factor (TF), the Brazilian ndHPFH mutation does not affect Sp1 binding. Thus, other TF may be involved in the reactivation of the A gamma globin gene in the Brazilian ndHPFH type. The aim of this study was to investigate the mechanism involved in the reactivation or repression of the A gamma globin gene in the Brazilian ndHPFH type and identify possible TF responsible for this phenotype. In vitro primary human erythroblast cultures, derived from human CD34+ hematopoietic cells from 4 Brazilian ndHPFH type subjects and 4 control subjects, were proliferated and differentiated into late stage erythroblasts. The nuclear extracts from predominantly basophilic and polychromatic erythroblasts were used to profile TF activity using Protein-DNA Array method. The analysis of the array densitometry identified a number of TF whose DNA binding activities were either enhanced or repressed in the Brazilian ndHPFH cultures. Among the TF analyzed, the NF-E1/YY1 and the PAX-1 were selected for this study. Since this assay requires a secondary method to confirm these results, nuclear extracts were used to conduct chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). ChIP was carried out using antibodies against NF-E1/YY1 and PAX-1 to quantify the binding to these TF to the –195 A gamma globin promoter region. EMSA was performed using probes with the same sequence spotted on the array membrane to analyze the activity of NF-E1/YY1 and PAX-1. Both methods confirmed and validated the previous array results. NF-E1/YY1 is a transcription factor that represses embryonic (epsilon) and fetal (gamma) globin genes. Protein-DNA array and EMSA showed a decreased binding of NF-E1/YY1 in Brazilian ndHPFH nuclear extracts and ChIP analysis revealed diminished NF-E1/YY1 occupancy at the –195 A gamma globin promoter region of Brazilian ndHPFH. The consensus binding site for NF-E1/YY1 is a CCAN motif that is observed between the –195 and –192 position in the A gamma globin promoter region. The C→G substitution at –195 position may disrupt this DNA binding site, cause decreased NF-E1/YY1 interaction and probably allows the binding of PAX-1, a transcriptional activator with a paired box DNA-binding domain that has as a DNA binding core motif, the sequence TTCCGC. This sequence, located between the –199 and –194 position in the A gamma globin promoter, is only presente in the Brazilian type of ndHPFH. Our protein-DNA array and EMSA results showed an increased binding of PAX-1 in the Brazilian ndHPFH nuclear extracts and quantitative ChIP analysis with anti-PAX-1 antibody showed that PAX-1 binds to the –195 A gamma globin promoter region only in the presence of this C→G substitution. These results suggest that the –195 site (C→G) in the A gamma globin promoter region may decrease NF-E1/YY1 binding and increase PAX-1 binding in this DNA region, probably resulting in the reactivation of the A gamma globin gene. The increase in the HbF levels in the Brazilian ndHPFH occurs differently from the British ndHPFH type and represents a novel mechanism of A gamma globin reactivation. Such findings may lead to the development of future therapeutic strategies for HbF induction in the treatment of other hemoglobinopathies. Support by FAPESP and CNPq. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5125 The TOB1 gene is a transcription factor responsible for the transduction of the gene ERBB2. It is a member of a family of cell suppressor proliferation proteins called TOB/BTG1 family; also, this gene operates on the inhibition of neoplastic transformation. The TOB1 gene presents a decreased expression in several types of cancer such as lung, breast, thyroid and stomach cancer. However, the function of this gene in chronic myeloid leukemia (CML) remains unknown. Aiming to evaluate the inhibition of gene TOB1 into BCR-ABL positive cells and trying to elucidate the molecular mechanisms associated with the inhibition of this gene in the CML we proceed to a more detailed study of this gene. The inhibition of this gene in K562 cells was performed using specific lentivirus. The effect of silencing TOB1 in the proliferation of K562 cells was assessed by the MTT assay after 48 hours of culture; in shTOB1 the proliferation was increased in comparison with shControl cells. To evaluate the synergistic effect between the inhibition of kinase tyrosine activity of BCR-ABL and the inhibition of TOB1 we performed a treatment with different concentrations of imatinib (0. 1, 0. 5 and 1μM), but we observed the decrease in cell proliferation of shTOB1 cells to similar levels of shControl cells only at the 1μM concentration. Therefore, the TOB1 silencing increased the proliferation of K562 cells without an additional effect of a treatment with Imatinib. To analyze the clonogenicity, we performed a formation of colonies assay, in methylcellulose, to determine whether silencing TOB1 could cause a change in the clonal growth of positive BCR-ABL cells. There was no significant change in the number of colonies that grew in cell culture shTOB1 compared to shControl cells. These results suggest that silencing TOB1 in K562 cells may not change the clonogenicity. In the assessment of cell cycle, the flow cytometry analysis revealed a significant accumulation of K562 cells in S phase, with consequent reduction of cells in the G2 phase of the cell cycle in cells shTOB1 compared to cells shControl. The TOB1 gene silencing in K562 cells kept the cells in the S phase and prevented the entry of cells in the G2 phase showing that the inhibition of gene TOB1 induced an increase in proliferation of K562 BCR-ABL cells. The level of apoptosis was assessed by flow cytometry after labeling the cells with anexin-V/PI. The Imatinib treatment presented dose-response in the induction of apoptosis as expected. However, a cumulative effect with TOB1 silencing was not observed. Furthermore, the apoptosis was also assessed by assays of caspases 3, 8 and 9, which showed an increase of the caspase activity of shControl cells in relation of the shTOB1 cells, showing that inhibition of this gene also changes the level of apoptosis. These results corroborate the literature data that report the relationship of this tumour suppressor gene in signalling pathways related to angiogenesis, carcinogenesis, apoptosis and metastasis. When we relate the results obtained with the LMC, we can consider the possibility of TOB1 regulation changes be related to modification of important signalling pathways such as AKT, PI3K, STAT3 and STAT5, among others. Furthermore, the inhibition of TOB1 may be related with an increase on the number of BCR-ABL positive cells and subsequent disease progression. In conclusion, this study confirmed literature data showing that TOB1 gene works as a tumour suppressor protein in cells of many types of cancer. From this work we can infer that in CML the expression of this gene is transformed, resulting in changing of the capacity of induction of apoptosis, decrease tumour necrosis and increase cell proliferation. This work was supported by FAPESP and INCT. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3195 The hereditary persistence of fetal hemoglobin (HPFH) is the consequence of impaired switching in adult life, which results in the continued expression of gamma globin gene. The Brazilian HPFH type is characterized by a C → G substitution at the –195 position of the A gamma globin gene promoter, and associated with HbF levels ranging from 6 to 16% in the heterozygote state. This study was undertaken to identify genes that may be involved in hemoglobin switching and/or maintenance of elevated HbF levels in Brazilian HPHF subjects. The Suppressive Subtractive Hybridization libraries were constructed using a pool of RNA extracted from reticulocytes of peripheral blood in individuals with normal hematological data and from subjects with Brazilian and 55 and 57 overexpressed genes were identified in the normal and Brazilian HPFH library, respectively; findings were validated by qRT-PCR and Western blotting. One transcription factor identified was FOXO3a, whose expression was increased in Brazilian HPFH subjects, compared to control subjects. Moreover, the non-phosphorylated Foxo3a protein that interacts with DNA, was detected only in Brazilian HPFH when analyzed by Western blotting. FOXO3a binds to PAX1 promoter, a transcription factor whose activity was higher in Brazilian HPFH. The FOXO3a/PAX1 complex may be important in the mechanism responsible for increasing HbF levels in this genetic disorder. Another gene analyzed was KLF1, a transcription factor known as a regulator of switching from the gamma to beta globin gene. This gene was underexpressed in the reticulocytes of HPFH subjects. Klf1 protein activity in erythroid cells of healthy donors was also increased compared to Brazilian HPFH, when analyzed by DNA/protein array. Results suggest that the decreased KLF1 levels in Brazilian HPFH favors the interaction between the A gamma globin gene and the Locus Control Region, agreeing with previous studies that demonstrated that knockdown of KLF1 in adult erythroid progenitors favors the formation of complexes that bind to the gamma globin gene promoter. MIER1 expression was also found to be decreased in Brazilian HPFH reticulocytes, compared to controls. The MIER1 gene is able to recruit chromatin remodeling-enzymes leading, to the formation of heterochromatin and, consequently, silencing genes. This MIER1 may be an important gene in gamma to beta globin gene switching, where it could help in the maintenance of a closed chromatin structure in the gamma globin gene in individuals with low HbF levels. The expression of the HOOK3 gene was decreased in Brazilian HPFH reticulocytes, compared to controls. The HOOK3 encodes a protein that interacts with a GTPase protein, stimulating INF-gamma, responsible for the phosphorylation of p65/p50 and RXR beta like proteins which regulate the transcription of the beta globin gene. The reduced beta globin gene expression in Brazilian HPFH resulting from the reactivation of gamma globin gene may be responsible for a decrease in the HOOK3 expression. These results suggest that, in the HPFH Brazilian type, the FOXO3a/PAX1 complex could contribute to the continued expression of the A gamma globin gene. Additionally, some cellular modifications, such as low Klf1 activity and decreased HOOK3 and MIER1 expression, could participate in the maintenance of HbF levels. These genes could be important in therapeutic approaches for the development of new HbF induction agents for the hemoglobinopathies. Support by FAPESP, CNPq.and INCTS Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4415 The CML is a clonal disease of stem cells and its main feature is the unregulated production of a tyrosine kinase protein called BCR-ABL, the progression of the disease to accelerated phase or blast crisis may be associated with genomic instability. Because of this, the use of tools for the study of gene expression could bring new insights in the understanding of these mechanisms in the CML. In a recent study using SSH libraries, we compared the gene expression pattern between granulocytes of health control and CML patients, and we identified the gene SEPT5 expressed only in CML patients. Although the studies in the literature, there is not a clear relationship between the expression of this gene and the development or progression of CML. SEPT5 is a member of nucleotide binding proteins called septins that were firstly described in yeast as cell division cycle regulatory proteins. This gene was reported in patients with AML translocated with MLL gene, in adult human brain and heart; it is also associated with alpha granules of human blood platelets. The aims of this study are to carry a functional analysis of SEPT5 in differents cells line and to study the relationship of this gene and the development and/or progression of CML. The gene expression evaluation was made in granulocytes, mononuclear cells and total leukocytes of CML patients and healthy blood donors in peripheral blood. It was also evaluated in bone marrow donors, in human cell lines (K562, HL60 and NB4) and in mice cell lines (BaF3/BCR-ABLp210 and BaF3T315I), performed by real-time PCR for the following genes: SEPT5, β-actin and GAPDH. Experiments were also performed to verify the difference between the chemotaxis of granulocytic cells from controls and patients by ELISA. Data were analysed statistically using the ANOVA followed by Dunnett’s test – P value of less than 0.05 was considered to be significant. The study was approved by the Research Ethic Committee of the Faculty of Medical Sciences of University of Campinas. The gene expression of SEPT5 was evaluated by real time PCR using the same samples used in the library construction to validate the results found in the SSH library. The data confirmed our previous results, showing that the SEPT5 expression is increased in all cells of patients compared to controls. The same results were observed when we studied the expression comparing individually patients and health blood donors, suggesting that this protein could be increased in all human cells that present the translocation BCR-ABL. The level of expression of this gene in HL60 and NB4 was significantly lower than in K562 cell line. The experiments with mice cell lines showed a higher expression of this gene in BaF3T315I when compared to BaF3BCR-ABLp210. We obtained a significant expression difference in all experiments (p