ALBERT

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: Introduction: Several studies reported combined contraceptives influence in hemostatic and lipid profile besides the concern about the thromboembolism and cardiovascular risks due to the steroids hormones use. As women with hemoglobin (Hb) variants have a pre-existing inflammatory condition and considering the high frequency of hemoglobin variant worldwide, this study aims to evaluate the association of hematological, lipid, glicemic, inflammatory and hemostatic profiles in women using combined oral contraceptives and carriers of hemoglobin variants. Methods: We performed a cross-sectional study including 591 women in reproductive-age. We investigated their hemoglobin profile and COCs use. Of them, we included 60 women with HbAA, 21 with HbAC, 25 with HbAS and 7 with HbSC profiles, all of them combined oral contraceptives users. Among those combined oral contraceptives nonusers, 9 were HbAC and 19 HbSC. We evaluated fasting serum glucose, triglycerides, total cholesterol, low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), C-reactive protein (CRP), fibrinogen, D-dimer and hematological profile among the studied groups. This study was conducted in accordance and approved by the Research Ethics Committee of the Fundação Oswaldo Cruz - FIOCRUZ, Brazil; and also with the Helsinki Declaration of 1975, and its revisions. Mann-Whitney U tests were used to compare two groups of values within the same variable. Results: We observed significant differences in some hematological and cardiometabolic parameters in women carriers of different Hb variants and using COCs. We found relevant increases in CRP levels in HbSC and HbAC women that seem to be associated with different types of progestins present in combined oral contraceptives formulations. Also, combined oral contraceptives use seems to be associated with decreased HDL-c levels in HbAC women. Otherwise, D-dimer levels were increased in all women with Hb variants, independently of the contraceptive use. Conclusions: Although combined oral contraceptive remains an important method to prevent unintended pregnancy, our data suggest that contraception in women carriers of Hb variants, including those in heterozygosis, should be carefully evaluated, especially, considering the pre-existent inflammatory and pro-thrombotic conditions that together with combined oral contraceptive use may result in additional health problems, such as cardiovascular and thromboembolic diseases. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Mouse models have allowed a wider investigation of the inflammatory process in Sickle cell disease (SCD). Previous studies have demonstrated that neutrophil recruitment inresponse to LPS is upregulated in Berkeley sickle mice and the development of acute LPS-induced airway inflammation is associated with enhanced expression of cytokines, chemokines and MMPs expression in lungs. MicroRNAs (miRNAs) belong to a regulatory class of RNAs with great importance in several biological processes, including the development and fate of immune cells, and in both innate and adaptive immune responses, whereby strong evidence links miRNAs expression to signalling pathways and receptors. Some miRNAs are positive regulators of inflammation and others are involved in the negative-feedback regulation of inflammatory processes. A growing amount of evidence indicates that these molecules are involved in various lung diseases. However, the expression profile of miRNAs in lung injury in SCD have not been investigated. Since pulmonary complications are an important cause of morbidity and mortality in SCD, the aim of this study was to determine whether miRNAs implicated in LPS-mediated inflammation are involved in neutrophil recruitment and inflammation in lung injury in SCD using the sickle mice model. Methods: Acute lung inflammation and injury was induced in wild- type (WT, C57BL/6, n=5) and Berkeley SCD mice (n=5) by intranasal administration of LPS (50 ml of 250 mg/ml). The vehicle group received a similar volume of sterile PBS. BAL was performed 4h after LPS challenge. qPCR analysis was used to examine gene expression and ELISA was used to determine protein production. For miRNAs expression analysis, extraction of lung RNA was performed using mirVanaTM miRNA Isolation kit and TaqManTM MicroRNA Reverse Transcription.The expression level of miRNAs was determined using the Taqman miRNA expression assay by qPCR with U6 snRNA as an endogenous control. Results: Levels of miR-15a-5p, miR-16-5p, miR-26a-5p and miR-98-5p in lung tissue increased almost twofold at 4h after LPS challenge in SS compared with SS PBS group (2.9, 3.7; 2.3; 2.9-fold change, respectively). The expressions of miR-106a-5p, miR-146a-5p, miR-146b-5p, and miR-181a-5p were not altered (2.0; 1.2; 1.55; 1.4-fold change, respectively). However, the expressions of miR-203a-3p and miR-223-3p were significantly up-regulated compared to animals from the PBS group (4.3 and 8.8 fold change, respectively, p

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: 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 2651 Background: LIGHT (TNFSF14; CD258), a recently-identified member of the TNF superfamily, is found associated with and produced from platelets, amongst other immune cells. Increased circulating levels of this protein have been observed in patients with myocardial infarction and acute atherothrombotic stroke and LIGHT has been proposed as a potential therapeutic target in atherosclerosis, due to its reported pro-thrombotic and pro-inflammatory effects upon human endothelial cells. This study evaluated whether production of LIGHT is altered in patients with sickle cell anemia (SCA), and the participation that the platelets (PLTs) may have in this production, since SCA is characterized by a significant chronic inflammation and endothelial activation that may initiate the vaso-occlusive process. Patients and Methods: Soluble LIGHT (sLIGHT) was determined in PLT-free plasma (obtained by sequential centrifugations and ultrafiltration) from healthy control individuals (CON), SCA patients in steady state (SCA) and SCA patients on hydroxyurea therapy (SCAHU; 20–30 mg/kg/day HU) by ELISA. Expressions of PLT-membrane LIGHT and PLT activation markers were evaluated by flow cytometry using anti-CD258-PE, anti-CD62P-FITC or anti-PAC1-FITC. Subjects had not taken ASA during the previous 14 days. Results: sLIGHT was significantly elevated in the plasma of SCA and SCAHU individuals, compared to CONs (SCA; 35.4 ± 9.4 pg/ml; SCAHU, 37.3 ± 7.5 pg/ml; CON, 9.7 ± 1.5 pg/ml, n=27, 27, 19, resp.; P

Description: Introduction Hereditary Persistence of Fetal Hemoglobin (HPFH) and δβ-thalassemia are genetic disorders characterized by elevated levels of fetal hemoglobin (HbF) in adulthood. Deletions of variable sizes, and at different positions, that involve the β-globin gene complex on chromosome 11 are associated with these disorders. The distinction between these conditions is made based on clinical and hematological data. Deletional HPFH includes a wide range of conditions, but typically it is characterized in heterozygotes by levels of HbF of 15% to 30% with normal red blood cell indices, while heterozygotes for δβ-thalassemia tend to have elevated levels of HbF that are lower (5% to 20%) and accompanied by mild anemia with hypochromic, microcytic red blood cell indices. MicroRNAs (miRNAs) have been described to have a possible role in globin switching and can modulate transcriptional erythroid-specific regulators. To the best of our knowledge miRNAs have not been analyzed in HPFH and δβ-thalassemia. The aim of this study was to investigate the miRNAs expression profile and possible post-transcriptional role of these molecules in relation to the lack of normal suppression of γ genes in these genetic disorders. Methods CD34(+)-derived erythroid cells from two HPFH-2 individuals and two δβ-thalassaemia Sicilian type patients (DB) and healthy controls (CTRL) were cultured for 13 days to examine the expression profile of miRNAs. The miRNAs were hybridized using an Agilent miRNA microarray platform and the profiles were obtained through bioinformatics data analysis using GeneSpring software. qPCR analysis was used to validate the miRNA expression (TaqMan® miRNAs assays) and to quantify gene expression of 19 transcription factors. Different databases, such as miRBase, TargetScan, microRNA.org and BioGPS were used to determine the predicted targets of miRNAs data found. Results Six miRNAs were up-regulated in HPFH and in DB compared to CTRL: miR-146b-5p, miR-181a, miR-342-3p, miR-362-3p, miR-362-5p and miR-365. Five miRNAs were up-regulated in HPFH, compared to DB: miR-223, miR-630, miR-638, miR-1246 and miR-1290. Nine miRNAs were up-regulated in DB compared to HPFH: miR-10a, miR-21*, miR-33b*, miR-96, miR-128, miR-194, miR-210, miR-424 and miR-1275. The ALK4 and the GATA2 mRNAs were significantly up-regulated in HPFH, compared to DB. The BCL11A and the SH3BGRL2 mRNAs were significantly down-regulated in HPFH compared to DB. In silico analysis and the literature show that several miRNAs have targets related to HbF production or erythropoiesis. These include miR-10a, miR-33b*, miR-96, miR-128, miR-210, miR-223, miR-342-3p, miR-362-3p, miR-424 and miR-630. Conclusion The comparison of miRNA and transcription factor profiles suggests differences in the expressions of several miRNAs that may influence γ-globin gene expression. These data may contribute to understanding the phenotypic differences found between deletional HPFH and δβ-thalassemia. Financial support by FAPESP and CNPq/INCTS. Disclosures: No relevant conflicts of interest to declare.

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: Chronic Graft versus Host disease (cGVHD) is a major limiting factor for the success of allo-HSCT. In this prospective study we aimed to evaluate the association between the kinetics of Regulatory T cells (TREG) and Conventional CD4+T cells (TCON) reconstitution and the emergence of cGVHD. We performed a detailed phenotypic analysis by multiparametric flow cytometry using fresh blood from 39 patients undergoing unrelated donor HSCT after a reduced intensity conditioning regimen containing ATG over a 2 year period, representing a total of 213 samples analyzed. GVHD prophylaxis consisted of cyclosporine plus mycophenolate mofetil. 11 patients were excluded due to disease relapse and/or death due to infection or acute GVHD in the first 9 months post-HSCT. We observed indiscriminately low numbers of TREG (CD4+CD127lowFoxp3+CD25bright) until mo 6 after HSCT and reduced TREG numbers in patients developing cGVHD (GVHD+) versus those who did not (GVHD-) (p=0.02 at mo 9). We further studied the dynamics of TREG subset reconstitution. CM TREG (CD45RA-CD62L+) was the predominant population in both patient groups. EM (CD45RA-CD62L-) was the second most abundant TREG subset. CM and EM TREG numbers were similar between patient groups. EMRA (CD45RA+CD62L-) TREG remained very low throughout the follow-up but were significantly increased at mo 9 in GVHD+ (p= 0.03). Interestingly, Naïve TREG (CD45RA+CD62L+CD95-) started to emerge at mo 9 and were significantly reduced in GVHD+ patients at mo 12 (0.71 vs 0.14 cells/µl; p=0.02) The Stem Cell Memory subset (SCM), identified as CD45RA+CD62L+CD95+, is thought to be self renewing and multipotent, being able to differentiate into CM, EM and TEMRA memory subsets. While in GVHD- SCM TREG started to emerge at mo 9, this subset remained low in GVHD+. Statistically significant differences were observed at mo 18 (0.91 vs 0.15 cells/µl; p=0.02). To ascertain the role of thymic output in TREG reconstitution we quantified CD31+ naïve TREG. Recent Thymic Emigrant cells (RTE) TREG were significantly reduced in GVHD+ at mo 12 (0.8 vs 0.16cells/µl; p=0.02) and at mo 18 (1.93 vs 0.28 cells/µl; p=0.02). In order to clarify whether both thymic output and peripheral expansion were contributing factors to decreased Naïve TREG, we quantified proliferation using Ki-67. TREG from GVHD+ patients proliferated less from months 2 to 18 reaching statistical significance at mo 9 and 12 (p= 0.003 and 0.02, respectively), suggesting that decreased TREG numbers are at least partly due to reduced peripheral expansion. Taken together, these observations suggest a compromised peripheral expansion and de novo generation of TREG through thymic output in cGVHD patients. Of note, susceptibility to apoptosis was not increased in TREG from GVHD+ patients, as Bcl-2 levels tended to be higher in relation to GVHD- patients. In the CD4+Foxp3- TCON population, we observed a clear predominance of EM in all patients. These cells, together with CM, are the first to appear at similar levels in both patient groups. EMRA emerged at higher numbers in GVHD+, although the results did not reach statistical significance. Naïve and RTE TCON started to emerge after mo 6 in GVHD-. Interestingly, these cells remained much lower in GVHD+ vs GVHD- patients throughout the follow-up, reaching statistical significance at mo 12 (p=0.03 for naïve; p=0.03 for RTE TCON). SCM TCON remained very low in all patients showing a tendency to be reduced in GVHD+ when compared to GVHD-. This difference reached statistical significance at mo 18 (6.85 vs 2.13 cells/µl; p=0.02). The small number of TREG and TCON subsets in GVHD+ patients were unlikely due to increased susceptibility to apoptosis, as assessed by Bcl-2 expression. The low numbers of cells within naïve and SCM TREG gates precluded the analysis of Bcl-2 expression in these subsets. Bcl-2 tended to be increased in total TREG CM and EM in GVHD+. In TCONs, Bcl-2 levels were similar in both patient groups. In summary, our data in patients developing cGVHD suggest that decreased thymic output and increased differentiation into terminally differentiated effector cells may have a negative impact on the number of naïve and SCM TCON and TREG. We speculate that the inability to generate and maintain the more immature TREG subsets may lead to decreased TREG numbers after 6 months post transplant, potentially resulting in decreased control of effector T cells contributing to the development of cGVHD. Disclosures Ritz: Kiadis: Membership on an entity's Board of Directors or advisory committees.

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.