Matheus Rodrigues Lopes

FMNL1 promotes proliferation and migration of leukemia cells

The human FMNL1 is expressed predominantly in hematopoietic cells and has been described previously as overexpressed in hematopoietic malignancies. However, it is not known whether FMNL1 contributes to leukemogenesis. Here, we investigate the FMNL1 function using two different human leukemia models: Namalwa and K562 cell lines. FMNL1 depletion reduced cell proliferation and colony formation in both leukemic cell types, as well as a decrease in the tumor growth of FMNL1‐depleted Namalwa cell xenografts. In addition, there was a decrease in migration and in TEM in FMNL1‐depleted Namalwa cells. FMNL1 endogenously associates with Rac1, and FMNL1 silencing resulted in an increased Rac1 activity. The reduced migration observed in FMNL1‐depleted cells was restored by inhibiting Rac activity. Our results indicate that FMNL1 stimulates leukemia cell proliferation as well as migration. This suggests that FMNL1 contributes to leukemogenesis and could act in part through Rac1 regulation.

Distinct expression profiles of MSI2 and NUMB genes in myelodysplastic syndromes and acute myeloid leukemia patients

Recent studies have indicated the Musashi2/NUMB pathway as the key regulator of differentiation in chronic myeloid leukemia; however, a comparison of both gene expressions has not yet been made in myelodysplastic syndrome (MDS) and in acute myeloid leukemia (AML). We herein, demonstrate a statistically significant down-modulation of NUMB expression level in high-risk MDS and AML, compared with control individuals. MSI2 expression was significantly reduced in low and high-risk MDS compared with normal control samples. NUMB expression was significantly lower than that of MSI2 in both MDS and AML patient samples, but no differences in the expression levels for either gene were observed in healthy bone marrow cells. Finally, NUMB expression was significantly up-regulated during differentiation of normal and low-risk MDS CD34(+) cells through the erythroid lineage. Taken together, results suggest the involvement of NUMB in MDS erythropoiesis; its down-modulation may have a role in MDS progression.

IL10 inversely correlates with the percentage of CD8 + cells in MDS patients

The role of the immune system in myelodysplastic syndrome (MDS) progression has been widely accepted, although mechanisms underlying this immune dysfunction are not clear. CD4(+) and CD8(+) lymphocyte profiles in the peripheral blood of MDS patients were evaluated and correlated with clinical characteristics, the expression of FOXP3 and the anti-inflammatory cytokines IL10, TGFβ1 and CTLA4. IL10 expression inversely correlated with the percentage of CD8(+) cells and was higher in high-risk MDS. Our findings provide further evidence for the role of T cell-mediated IL10 production in MDS and strengthen the idea of distinct cytokine profiles in low and high-risk MDS.

Molecular effects of the phosphatidylinositol-3-kinase inhibitor NVP-BKM120 on T and B-cell acute lymphoblastic leukaemia.

BACKGROUND Constitutive activation of the PI3K pathway in T cell acute lymphoblastic leukaemia (T-ALL) has been reported and in a mouse model, PI3K activation, together with MYC, cooperates in Burkitt lymphoma (BL) pathogenesis. We investigated the effects of NVP-BKM120, a potent pan-class I PI3K inhibitor, in lymphoblastic leukaemia cell lines. METHODS Effects of NVP-BKM120 on cell viability, clonogenicity, apoptosis, cell cycle, cell signalling and autophagy were assessed in vitro on T-ALL (Jurkat and MOLT-4) and BL (Daudi and NAMALWA) cell lines. RESULTS NVP-BKM120 treatment decreased cell viability and clonogenic growth in all tested cells. Moreover, the drug arrested cell cycling in association with a decrease in Cyclin B1 protein levels, and increased apoptosis. Immunoblotting analysis of cells treated with the drug revealed decreased phosphorylation, in a dose-dependent manner, of AKT, mTOR, P70S6K and 4EBP1, with stable total protein levels. Additionally, we observed a dose-dependent decrease in BAD phosphorylation, in association with augmented BAX:BCL2 ratio. Quantification of autophagy showed a dose-dependent increase in acidic vesicular organelles in all cells tested. CONCLUSION In summary, our present study establishes that NVP-BKM120 presents an effective antitumour activity against T-ALL and BL cell lines.

Abnormal Hedgehog pathway in myelodysplastic syndrome and its impact on patients’ outcome

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and by cytopenias. Approximately 30% of patients with MDS progress to acute myeloid leukemia.[1][1] Thus, it is important to identify risk factors for AML progression and to guide

Serine Protease Inhibitor Kunitz-Type 2 Is Downregulated in Myelodysplastic Syndromes and Modulates Cell–Cell Adhesion

Myelodysplastic syndromes (MDS) are clonal disorders involving hematopoietic stem cells (HSC) characterized by ineffective hematopoiesis. In addition to HSC defects, a defective hematopoiesis supporting capacity of mesenchymal stromal cells (MSCs) in the microenvironment niche has been implicated in MDS pathophysiology. The interaction between the dysfunctional MSCs MDS and HSC regulates diverse adhesion-related processes, such as progenitor cell survival, proliferation, differentiation, and self-renewal. As previously reported, a microarray analysis identified serine protease inhibitor kunitz-type 2 (SPINT2), an inhibitor of hepatocyte growth factor (HGF) activation, to be downregulated in MSCs from MDS patients. To define the role of SPINT2 in MDS hematopoietic microenvironment, an analysis of the effect of SPINT2 silencing in MSCs was carried out. We herein reported significantly lower levels of SPINT2 whereas HGF was expressed at higher levels in MSCs from MDS patients compared with healthy controls. SPINT2 underexpression results in an increased expression, production, and secretion of HGF and stromal cell-derived factor 1 (SDF-1) by MSCs. An increased adhesion of normal HSC or malignant cells onto MSCs silenced for SPINT2 was also observed. The altered MSCs adhesion in SPINT2-knockdown cells was correlated with increased CD49b and CD49d expression and with a decrease in CD49e expression. Our results suggest that the SPINT2 underexpression in the MSC from MDS patients is probably involved in the adhesion of progenitors to the bone marrow niche, through an increased HGF and SDF-1 signaling pathway.

De novo AML exhibits greater microenvironment dysregulation compared to AML with myelodysplasia-related changes

The interaction between the bone marrow microenvironment and malignant hematopoietic cells can result in the protection of leukemia cells from chemotherapy in both myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). We, herein, characterized the changes in cytokine expression and the function of mesenchymal stromal cells (MSC) in patients with MDS, AML with myelodysplasia-related changes (MRC), a well-recognized clinical subtype of secondary AML, and de novo AML. We observed a significant inhibitory effect of MDS-MSC on T lymphocyte proliferation and no significant differences in any of the cytokines tested. AML-MSC inhibited T-cell proliferation only at a very low MSC/T cell ratio. When compared to the control, AML-MRCderived MSC presented a significant increase in IL6 expression, whereas de novo AML MSC presented a significant increase in the expression levels of VEGFA, CXCL12, RPGE2, IDO, IL1β, IL6 and IL32, followed by a decrease in IL10 expression. Furthermore, data indicate that IL-32 regulates stromal cell proliferation, has a chemotactic potential and participates in stromal cell crosstalk with leukemia cells, which could result in chemoresistance. Our results suggest that the differences between AML-MRC and de novo AML also extend into the leukemic stem cell niche and that IL-32 can participate in the regulation of the bone marrow cytokine milieu.

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 patients 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.

Descriptive study of the prevalence of anemia, hypertension, diabetes and quality of life in a randomly selected population of elderly subjects from São Paulo

Background The rapid increase in the aged population has resulted in a growing number of cases of chronic diseases. This increase is an important demographic change that low- and middle-income countries have to face and poses a new challenge to health services. One of the first steps to formulate public policies is to understand the reality of each countrys aging population. This study describes the prevalence of anemia, hypertension and diabetes and the overall health status in pre-elderly and elderly subjects enrolled in two primary health care clinics, Eldorado and Piraporinha, in the city of Diadema, São Paulo. Method A cross-sectional study was conducted with 373 participants. Clinical data were collected from patient charts and the degree of disability and common mental disorders, as well as demographic data were obtained by interviews. Results The prevalence of anemia was approximately 11% and hypertension was 70% and 81% in Eldorado and Piraporinha, respectively. The frequency of diabetes was 52% in Eldorado and 30% in Piraporinha. The subjects of both health care clinics reported having difficulties in some of their daily physical and instrumental activities, with physical symptoms and emotional disorders. Conclusion Anemia, hypertension and diabetes are prevalent in the studied population, and patients showed degrees of dependency and impaired health status.

IL32 expression in peripheral blood CD3+ cells from myelodysplastic syndromes patients

BackgroundMyelodysplastic syndromes (MDS) are a heterogeneous group of disorders characterized by ineffective hematopoiesis and risk of leukemia transformation. There is evidence to suggest the participation of immune system deregulation in MDS pathogenesis. Interleukin-32 (IL-32) is a newly described multifunctional cytokine reported as an important mediator in autoimmune and inflammatory disorders. In the present study, we reported the expression of IL32 and IL32 transcript variants (α, β, γ and δ) in peripheral blood CD3+ cells from healthy controls and MDS patients.MethodsCD3+ cells were isolated by immunomagnetic cell sorting from thirty-nine untreated MDS patients and twenty-nine healthy donors. Gene expression was evaluated by quantitative PCR. For statistical analysis, Mann–Whitney test, Kruskal-Wallis test with Dunns post test and Log-rank (Mantel-Cox) were used, as appropriate. A p value <0.05 was considered statistically significant.ResultsIL32 expression and IL32 transcript variants IL32α, IL32β, IL32γ, and IL32δ, were similar in peripheral blood CD3+ cells from healthy donors and MDS patients. Increased IL-32α expression was an independent predictor for MDS disease progression by univariate and multivariate analysis.ConclusionsWe observed that IL32 expression is not differently expressed in CD3+ cells from MDS patients; nevertheless IL32α has a potential role in disease progression.