ALBERT

Description: Abstract 1482 The crosstalk between leukemic cells and their microenvironment provides pivotal signals for the localization and progression of leukemias. The stromal derived factor-1 (SDF-1)/CXCR4 pathway is deregulated in hematology malignances, such as acute lymphoblastic leukemia (ALL). SDF-1 represents the major chemokine for initiating stem cell chemotactic migration. The majority of cytokines that mediate stem cell chemotaxis does so via modulation either of SDF-1 or of its receptor, CXCR4. CXCR4 receptor is expressed in leukemic cells enabling cells to access marrow niches that normally are restricted to quiescent stem cells, thereby ensuring its protection of cell death resulting in a worse prognosis. Recently, CXCR7 was identified as another SDF-1-binding receptor, but its contribution to SDF-1 – mediated effects in hematopoietic cells is still poorly explored, even though the CXCR7 relationship with tumor progression in non-hematopoietic malignancies is well established. Given that SDF-1/CXCR4 signaling is altered in patients with leukemia and that there is little information regarding CXCR7 in leukemia we investigated its expression in patients and leukemia cell lines. In addition, the relationship of CXCR4 and CXCR7 in potentiating the SDF-1 response was also investigated. mRNA expression of CXCR7 was analyzed by Real-time PCR (normalized by GAPDH and HPRT) in bone marrow samples of 29 acute myeloid leukemia (AML), 11 ALL patients and 12 control subjects (healthy donors). CXCR7 protein expression in myeloid cell lines (U937, P39, K562 and KG -1) and lymphoblastic cell lines (Jurkat, Molt-4, Raji e Daudi) was analyzed by western blot. Localization of CXCR4 and CXCR7 proteins was investigated using flow cytometry and confocal microscopic analysis and CXCR7 knockdown cells were obtained by transduction with lentivirus-mediated shRNA. These cells were treated with AMD3100 (antagonist CXCR4) and their chemotactic capacity was analyzed by transwell chemotaxis assay. CXCR7 was significantly higher expressed in ALL compared to AML and control subjects (p=0.0008, Mann-Whitney test). CXCR7 protein (western blot with ABCAM antibody) was also higher expressed in lymphoblastic cell lines (Molt-4 and Jurkat) compared with myeloid cells. The subcellular location of CXCR7 and CXCR4 by confocal microscopy and flow cytometry evidenced CXCR7 in the membrane of Molt-4 cells and in the cytoplasm of Jurkat cells whereas CXCR4 was in the membrane of both cell lines. Interestingly, we also noticed that, after SDF-1 induction, Molt-4 cells have higher chemotactic ability compared with Jurkat (median Molt 4=52.0 ± 5 vs Jurkat=24.1 ± 3, p=0.0079, Mann-Whitney test) which may be related with the membrane availability of CXCR7. In addition, the inhibition of CXCR7 or CXCR4 resulted in significant changes in Molt4 and Jurkat chemotactic response (0.01〉p〈 0.02, Mann-Whitney test), however, the inhibition of both CXCR7 and CXCR4 resulted in a more significant reduction in cell migration (p=0.0079/Molt-4; p=0.0043/Jurkat, Mann-Whitney test). Increased expression of CXCR7, as here observed in lymphoblastic leukemia cells, is a phenomenon already described in a variety of solid tumor cell lines such as brain, prostate and lung. In solid tumors, CXCR7 mainly increases the proliferation of malignant cells. These results suggest that the biological function of CXCR7 depends on its tissue and organ localization and that, in acute lymphoblastic leukemia may have a role in cell chemotaxis, potentiating CXCR4 response to SDF-1 and thus, could contribute for leukemia initiating cell recruitment to niches once occupied by normal hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3813 The pathogenesis of MDS is complex and remains elusive. The molecular characterization of this disease has been a tool to obtain a better understanding. Our group recently identified new possible target genes involved in MDS pathophysiology through the microarray analysis of CD34+ cells from MDS patients. One of the interesting overexpressed genes found was the HCK gene, a Src kinase family member. This family integrates signaling from a variety of cell-surface receptors, such as RTK, EPO receptor, chemokine receptor, MET and CXCR4, to regulate diverse cellular responses including proliferation, differentiation, apoptosis and migration. HCK is restrictedly expressed in hematopoietic cells and overexpressed in some types of solid cancer and in acute lymphoid leukemia; however, functional role of HCK in MDS and myeloid cells is still unknown. Here, using qPCR we investigated the HCK mRNA expression in CD34+ and total bone marrow (BM) cells from 54 MDS patients (FAB 39 low-risk and 15 high-risk), receiving no treatment, and 24 normal donors. We evaluated the role of HCK, studying its effect on proliferation, cell cycle and apoptosis by silencing this gene in U937 myeloid cell line. To inhibit HCK, specific shRNA expressing lentiviral vectors targeting the HCK gene or no specific sequence were used. Cell growth was measured by MTT colorimetric assay. Annexin-PI was used to evaluate apoptosis and cell cycle was analyzed by flow cytometry. These assays were carried out in lentiviral transduced cells treated, or not, with rapamycin(10 or 100nM) and LY294002(50nM). We further analyzed the HCK expression during granulocytic and erythroid differentiation of established leukemia cell lines models. Granulocytic differentiation was induced in NB4 or HL60 cells by treatment with ATRA(10−6 M) for 4 days. Erythroid differentiation was induced in KU812 cells by treatment with HE(50μM) and HU(100μM) for 4 days. We observed a significant increase in HCK mRNA expression of MDS CD34+ cells (P=0.002) and BM cells (P=0.03) compared to normal cells. HCK mRNA expression in low-risk MDS cells was higher compared to high-risk MDS cells. In U937 cells, HCK inhibition resulted in a significant decrease in proliferation compared to control cells (P

Description: Abstract 3824 The factor SDF-1 (stromal derived factor-1) was identified as an important chemoattractant factor produced by bone marrow cells. SDF-1 acts on its receptor CXCR4 and plays primordial function in migration, retention and development of hematopoietic progenitors in bone marrow. CXCR4 is expressed in leukemic cells and enables them to access marrow niches that normally are restricted to quiescent stem cells, thereby ensuring its protection from cell death resulting in a worse prognosis. In addition, it may induce activity of metalloproteinases (MMPs), which are enzymes that digest the extracellular matrix, making tumor cells more infiltrating. Moreover, higher levels of TIMP-2, an inhibitor of metalloproteinase 2 (MMP2), correlates with better prognosis in solid tumors. Recently, CXCR7 was identified as another SDF-1-binding receptor and the involvement of CXCR7 with tumor progression is well established in non-hematopoietic malignancies. Since it is well established that CD34 + progenitor cells from patients with myelodysplastic syndromes (MDS) are not attracted by gradient of SDF-1 despite of having CXCR4 normal expression, we addressed if MDS cells have an abnormal localization of CXCR4 or association with CXCR7. P39 and U937 cell line were used as a model of MDS and AML, respectively. Western blot analysis showed similar expression levels of CXCR4 and CXCR7 in both cell lines however we found, by confocal microscopy and flow cytometry, that CXCR4 was localized in the cytoplasm) of P39 cells while it was was in the membrane of U937 cells. Since the protein quinase C (PKC z) is related to the SDF-1/CXCR4 signaling by increasing CXCR4 expression and its membrane availability, we overexpressed HA-tagged PKCz in P39 cells.This procedure resulted in translocation of CXCR4 to the membrane of P39 cells but did not change the CXCR7 subcellular localization. Transwell chemotaxis assay showed that P39 cells overexpressing PKCz displayed higher chemotactic ability upon SDF-1 treatment compared with control P39 (35 fold increase pcDNA3-PKCz-HA vs pcDNA3-HA transfected P39 cells, p=0.0032; x2 test), suggesting that PKCz restored the chemotactic capacity of P39 cells. RNA expression of CXCR7 and TIMP-2 was analyzed by Real-time PCR (normalized by GAPDH and HPRT) in bone marrow samples from 50 MDS (FAB= 22 RA, 8 RARS, 20 RAEB), 29 acute myeloid leukemia (AML) patients and 11 healthy donors. CXCR7 mRNA expression did not differ significantly comparing all groups: controls, MDS (low or high risk) and AML whereas the expression of TIMP- 2 mRNA was significantly decreased in high risk MDS (p=0.0033) and AML (p=0.0003), (Mann-Whitney test) compared with normal controls. Taken together, our results suggest that a defect in the PKC ζ/CXCR4 pathway is involved with the unresponsiveness of MDS cells to SDF-1 and TIMP-2 downregulation could play a role in worse prognosis of myeloid malignancies. The role of CXCR7 is still undefined in myeloid malignancies. Disclosures: No relevant conflicts of interest to declare.

Description: Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells (HSC) characterized by ineffective hematopoiesis. In addition to HSC defects, an important role is also played by the hematopoietic microenvironment niche that has as key component the mesenchymal stromal cells (MSC). The MSC of MDS patients have morphological and functional abnormalities. Recently, our group identified new possible target genes involved in MDS pathophysiology through microarray analysis of MSC from MDS patients. An interesting underexpressed gene found was SPINT2, a gene that encodes a transmembrane protein which inhibits the hepatocyte growth factor activator (HGFA), the enzyme responsible for the conversion of hepatocyte growth factor (HGF) into its active form. SPINT2 is downregulated in some types of solid cancer and correlated with their prognostic and progression; however, the functional role of SPINT2 in MDS remains unknown. We herein investigated the role of SPINT2 in MSC, studying HGF and SDF1 secretion and cell adhesion with normal CD34+, P39 and U937 cells by silencing SPINT2 gene in HS5 and HS-27a stromal cell lines. We also investigated, using qPCR, SPINT2 and HGF mRNA expression in MSC and total bone marrow (BM) cells from 56 untreated MDS patients (WHO 35 low-risk, 21 high-risk) and 28 healthy donors. To inhibit SPINT2, specific shRNA expressing lentiviral vectors targeting SPINT2 gene or no specific sequence were used. The HGF and SDF1 secretion in cell supernatant from the cells silenced or not for SPINT2 was measured by BioPlex after 6, 12, 24 and 48h. The cell-cell adhesion of CD34+, P39 or U937 cells onto transduced stromal cells and the adhesion molecule profile were analyzed by flow cytometry. We observed a significant decrease in SPINT2 mRNA expression of MDS MSC (P=0.006) and MDS BM cells (P=0.03) compared to normal cells. Further, HGF mRNA expression of MDS MSC was significantly increased (P=0.01) compared to normal cells. Spearman analysis showed a negative significant correlation between SPINT2 and HGF expressions (P=0.01;r2=0.60). In both stromal cell lines, SPINT2 inhibition resulted in a significant increase in HGF secretion after 24 and 48h and a significant increased secretion of SDF1 after 48h. Moreover, SPINT2 silencing induced a significant increased adherence of CD34+, P39 and U937 cells onto stromal cells probably due to the alteration in integrin expression, since an increase in CD49b and CD49d and decrease in CD49e expressions were also observed in cells silenced for SPINT2. Considering that SPINT2 limits signaling via HGF pathway by inhibiting HGF activation through HGFA, the SPINT2 underexpression in MDS would allow the conversion of the inactive HGF monomer to an active heterodimer. Interestingly, a significant decrease of SPINT2 and increase of HGF expressions were observed in MSC of MDS patients, compared to normal cells. Moreover, recent studies have demonstrated that HGF serum levels are significantly increased in MDS patients and dependent on MDS severity. In addition, SPINT2 inhibition in stromal cells significantly increased HGF secretion by these cells. The increased HGF secretion can result in an autocrine regulation which induces the production and secretion of SDF1 by stromal cells themselves. Cytokines secretions provided by MSC are required for adhesion, survival and proliferation of HSC cells. In this way, in MDS MSC, SPINT2 underexpression and, consequently, increased HGF and SDF1 secretion, may lead to an increased adhesion between MSC and normal or malignant HSC cells. Corroborating our hypotheses, we found overexpression of integrins CD49b and CD49d, which mediate cell-MSC interaction, and a decrease in CD49e expression, an integrin that promotes interaction with extracellular matrix. The interaction onto MSC contributes to the maintenance of the stem and malignant cell properties, such as self-renewal, survival and proliferation. Cytokine secretion and cell adhesion onto MSC is important for MDS physiology. Hence, we demonstrate for the first time that, in MDS, SPINT2 plays a role in the HGF and SDF1 secretion by MSC, resulting in an alteration in cell-cell adhesion and molecule adhesion profile. In view of these data, the SPINT2 expression alteration in MDS MSC may constitute a particular mechanism of MDS pathophysiology and maintenance of self-renewal, homing and proliferation of HSC and malignant clones in MDS.Support: FAPESP, CNPq Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: The advent of whole transcriptome sequencing technologies revealed that most of the human genome is transcribed, producing a large repertoire of long noncoding RNAs (lncRNAs). They are known to participate in epigenetic regulation at different levels and alter mRNA stability of coding genes, emerging as key players in carcinogenesis. In the present study, we identified a novel unspliced long noncoding RNA that is transcribed from the opposite strand on the NR4A1 gene locus. The tumor suppressor NR4A1 has been associated with the regulation of apoptosis and proliferation in various tumors and shows a reduced expression in myelodysplastic syndrome (MDS). Additionally, the abrogation of this locus in murine models caused the development of acute myeloid leukemia (AML). We hypothesized that lncNR4A1 plays an important role in hematopoietic malignancy pathogenesis and possibly regulates its coding counterpart. Methods: Expression of lncNR4A1 and its sense gene NR4A1 were evaluated in CD34+ cells from bone marrow of normal donors (n=7) and patients (n= 27; MDS= 14 [low‐risk=8, high‐risk=6], AML with myelodysplastic related changes [AML‐MRC]=4 and de novo AML=9) by qRT-PCR. Results were expressed as mean (minimum-maximum) after appropriate statistical analysis. The orientation and full-length of lncNR4A1 were confirmed by strandspecific RT-PCR, RACE-PCR and sequencing techniques. For functional analysis, lncNR4A1 was inhibited with a lentiviral vector system in U937 cells. Apoptosis was evaluated by Annexin‐V/PI and the cell cycle analysis was performed applying PI incorporation by flow cytometer analysis. Mitochondrial activity was evaluated with MTT assays. Western blot was used to measure the NR4A1 protein expression and for monitoring key proteins in the cell cycle signaling pathway. Results: NR4A1 mRNA was significantly decreased in bone marrow/CD34+ from patients with MDS and AML compared to healthy donors (p