Bruna Palodetto

MDR-1 and GST polymorphisms are involved in myelodysplasia progression.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell disorders characterized by abnormal hematopoietic differentiation and maturation, which progress toward acute leukemia in approximately 30% of the cases. Drug metabolism polymorphisms in Cytochrome P450 2B6 (CYP2B6), Glutathione S-transferase (GST) and Dehydrogenase Quinone 1 (NQO1) enzymes and P-glycoprotein (MDR-1) could modify enzyme activity. Thus, the aim of this study was to identify the influence of CYP2B6 G15631T, GSTT1, GSTM1, NQO1 C609T and MDR-1 C3435T polymorphisms on MDS progression. We analyzed 78 MDS patients using the PCR-RFLP and multiplex method. The frequency of GST deletions and MDR-1 CC genotype was lower in progression-free patients compared to patients with progression; GST: 17% vs. 35% (P=0.018); MDR-1 gene: 19% vs. 48% (P=0.012). We also verified the influence of GST deletions and MDR-1 C3435T on patient overall survival and found no significant difference (RR=0.75; P=0.599 and RR=0.79; P=0.594 respectively). We concluded that GSTM1 deletion may contribute toward MDS progression probably due to toxic metabolite accumulation which generates cell toxicity and DNA damage. Moreover, MDR-1 C3435T may have a protective effect against MDS progression because the expected lower expression of P-glycoprotein would lead to a higher degree of cell death. To the best of our knowledge, this is the first study showing the relationship of these polymorphisms with MDS progression.

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.

Hematopoietic cell kinase (HCK) is a potential therapeutic target for dysplastic and leukemic cells due to integration of erythropoietin/PI3K pathway and regulation of erythropoiesis: HCK in erythropoietin/PI3K pathway

New drug development for neoplasm treatment is nowadays based on molecular targets that participate in the disease pathogenesis and tumor phenotype. Herein, we describe a new specific pharmacological hematopoietic cell kinase (HCK) inhibitor (iHCK-37) that was able to reduce PI3K/AKT and MAPK/ERK pathways activation after erythropoietin induction in cells with high HCK expression: iHCK-37 treatment increased leukemic cells death and, very importantly, did not affect normal hematopoietic stem cells. We also present evidence that HCK, one of Src kinase family (SFK) member, regulates early-stage erythroid cell differentiation by acting as an upstream target of a frequently deregulated pathway in hematologic neoplasms, PI3K/AKT and MAPK/ERK. Notably, HCK levels were highly increased in stem cells from patients with some diseases, as Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML), that are associated with ineffective erythropoiesis These discoveries support the exploration of the new pharmacological iHCK-37 in future preclinical and clinical studies.

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.